Combinational compositions and methods for treatment of cancer

ABSTRACT

The present invention provides methods of treating a cell proliferative disorder, such as a cancer, by administering to a subject in need thereof a therapeutically effective amount of a pyrroloquinolinyl-pyrrole-2,5-dione compound or a pyrroloquinolinyl-pyrrolidine-2,5-dione compound in combination with a therapeutically effective amount of a second anti-proliferative agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/152,138, filed Feb. 12, 2009 and U.S. Provisional Application No.61/170,471, filed Apr. 17, 2009. The contents of each of theseapplications are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States,exceeded only by heart disease. (Cancer Facts and Figures 2004, AmericanCancer Society, Inc.) Despite recent advances in cancer diagnosis andtreatment, surgery and radiotherapy may be curative if a cancer is foundearly, but current drug therapies for metastatic disease are mostlypalliative and seldom offer a long-term cure. Even with newchemotherapies entering the market, the need continues for new drugseffective in monotherapy or in combination with existing agents as firstline therapy, and as second and third line therapies in treatment ofresistant tumors.

Cancer cells are by definition heterogeneous. For example, within asingle tissue or cell type, multiple mutational ‘mechanisms’ may lead tothe development of cancer. As such, heterogeneity frequently existsbetween cancer cells taken from tumors of the same tissue and samehistiotype that have originated in different individuals. Frequentlyobserved mutational ‘mechanisms’ associated with some cancers may differbetween one tissue type and another (e.g., frequently observedmutational ‘mechanisms’ leading to colon cancer may differ fromfrequently observed ‘mechanisms’ leading to leukemias). It is thereforeoften difficult to predict whether a particular cancer will respond to aparticular chemotherapeutic agent. (Cancer Medicine, 5th Edition, Bastet al. eds., B.C. Decker Inc., Hamilton, Ontario)

Breast cancer is the most frequently diagnosed non-skin cancer in women,and ranks second among cancer deaths in women, after lung cancer.(Cancer Facts and Figures 2004, American Cancer Society, Inc.) Currenttreatment options for breast cancer include surgery, radiotherapy, andchemotherapy/hormone therapy with agents such as tamoxifen, aromataseinhibitors, HERCEPTIN® (trastuzumab), TAXOL® (paclitaxel),cyclophosphamide, methotrexate, doxorubicin (Adriamycin®), and5-fluorouracil (5-FU). Despite improvements in cancer diagnostics andtherapeutics, breast cancer incidence rates have continued to increasesince 1980. In 2004, about 215,000 new cases of breast cancer areexpected in women, and about 1,450 new cases of breast cancer areexpected in men. Id. Accordingly, new compounds and methods for treatingbreast cancer are needed.

Components of cellular signal transduction pathways that regulate thegrowth and differentiation of normal cells can, when dysregulated, leadto the development of cellular proliferative disorders and cancer.Mutations in cellular signaling proteins may cause such proteins tobecome expressed or activated at inappropriate levels or atinappropriate times during the cell cycle, which in turn may lead touncontrolled cellular growth or changes in cell-cell attachmentproperties. For example, dysregulation of receptor tyrosine kinases bymutation, gene rearrangement, gene amplification, and overexpression ofboth receptor and ligand has been implicated in the development andprogression of human cancers.

The c-Met receptor tyrosine kinase is the only known high-affinityreceptor for hepatocyte growth factor (HGF), also known as scatterfactor. Binding of HGF to the c-Met extracellular ligand-binding domainresults in receptor multimerization and phosphorylation of multipletyrosine residues in the intracellular portion of c-Met. Activation ofc-Met results in the binding and phosphorylation of adaptor proteinssuch as Gab-1, Grb-2, Shc, and c-Cbl, and subsequent activation ofsignal transducers such as PI3K, PLC-γ, STATs, ERK1 and 2 and FAK. c-Metand HGF are dysregulated in human cancers, and may contribute todysregulation of cell growth, tumor cell dissemination, and tumorinvasion during disease progression and metastasis. (See, e.g., Journalof Clinical Investigation 109: 863-867 (2002) and Cancer Cell pp 5-6Jul. 2004) c-Met and HGF are highly expressed relative to surroundingtissue in numerous cancers, and their expression correlates with poorpatient prognosis. (See, e.g., Journal of Cellular Biochemistry 86:665-677 (2002); Int. J. Cancer (Pred. Oncol.) 74: 301-309 (1997);Clinical Cancer Research 9: 1480-1488 (2003); and Cancer Research 62:589-596 (2002)) Without intending to be bound by theory, c-Met and HGFmay protect tumors against cell death induced by DNA damaging agents,and as such may contribute to chemoresistance and radioresistance oftumors. Without intending to be limited by any theory, inhibitors ofc-Met may be useful as therapeutic agents in the treatment ofproliferative disorders including breast cancer. (See, e.g., Cancer andMetastasis Reviews 22: 309-325 (2003))

The references cited herein are not admitted to be prior art to theclaimed invention.

SUMMARY OF THE INVENTION

The present invention provides a method of treating a cell proliferativedisorder, the method comprising administering to a subject in needthereof a therapeutically effective amount of a compound of formula III,IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable saltthereof, or a prodrug or metabolite thereof, with one or morepharmaceutically acceptable carriers or excipients, alone, or incombination with a therapeutically effective amount of a secondanti-proliferative agent, with one or more pharmaceutically acceptablecarriers or excipients, wherein the cell proliferation disorder istreated.

The compound of formula III, IIIa, IVa, IVb, Va, or Vb can be(+)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,(−)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,(+)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,or(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Preferably, the compound is(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

The second anti-proliferative agent can be a a kinase inhibitor, analkylating agent, an antibiotic, an anti-metabolite, a detoxifyingagent, an interferon, a polyclonal or monoclonal antibody, a HER2inhibitor, a histone deacetylase inhibitor, a hormone, a mitoticinhibitor, an MTOR inhibitor, a taxane or taxane derivative, anaromatase inhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, or a cytidine analogue drug. Preferably, thekinase inhibitor is serine/threonine kinase inhibitor or a tyrosinekinase inhibitor. Preferred kinase inhibitors include, but are notlimited to, sorafenib, sunitinib, erlotinib, imatinib, and gefitinib.Preferred alkylating agents include, but are not limited to, cisplatinor carboplatin. Preferred anti-metabolites include, but are not limitedto, gemcitabine, fluorouracil (5-FU), TS-1 or capecitabine. Preferredmitotic inhibitors include, but are not limited to, camptothecin oririnotecan. Preferred taxane or taxane derivatives include, but are notlimited to, paclitaxel or docetaxel.

The cell proliferative disorder can be a precancerous condition orcancer. The cell proliferative disorder can be a hematologic tumor ormalignancy, or a solid tumor (or tumors). The methods of treating cancerinclude a reduction in tumor size. Alternatively, or in addition, thecancer is metastatic cancer and this method of treatment includesinhibition of metastatic cancer cell invasion. The method can furtherinclude radiation therapy. The cancer can be lung cancer, small celllung cancer, non-small cell lung cancer (NSCLC), colon cancer, breastcancer, pancreatic cancer, prostate cancer, renal cancer, cervicalcancer, brain cancer, gastric/stomach cancer, uterine cancer, intestinalcancer, hepatic cancer, chronic myelogenous leukemia, melanoma, ovariancancer, translocation-associated renal cell carcinoma (RCC), alveolarsoft part sarcoma (ASPS), clear cell sarcoma (CCS), or hepatocellularcarcinoma (HCC).

Cells with a proliferative disorder can contain DNA encoding c-Met.Alternatively, or in addition, cells with a proliferative disorder havea constitutively enhanced c-Met activity. Preferably, the cellproliferative disease is cancer, and particularly those cancers whichexpress c-Met at high levels or express active c-Met. Thus, the presentinvention provides a method of treating cell proliferative disorderswhere the cells express c-Met at high levels or express active c-Met.The present invention further provides a method of treating a cellproliferative disorder comprising selectively modulating an activity ofc-Met, without significantly inhibiting the activity of Protein KinaseC.

Preferably, the subject is a mammal. More preferably, the subject is ahuman.

Preferably, the compound of formula III, IIIa, IVa, IVb, Va, or Vb ofthe methods described herein is(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand is administered at a dose of 360 mg, provided twice a day.Alternatively, the composition is administered at a maximal daily doseof 720 mg.

Preferably, the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand the second anti-proliferative agent are administered intravenously,orally or intraperitoneally. The second anti-proliferative agent can beadministered simultaneously with, preceding administration of, orfollowing administration of the composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Preferably, the second anti-proliferative agent is administered within24 hours after the composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered.

The present invention also provides a kit for the treatment of a cellproliferative disorder in a subject comprising separate vials containinga composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,or a pharmaceutically acceptable salt thereof, or a prodrug ormetabolite thereof, and a second anti-proliferative agent, withinstructions for administering said composition and secondanti-proliferative agent.

Preferably, the subject is a mammal. More preferably, the subject is ahuman.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 sets forth the chemical structures of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

FIG. 2A-B is a depiction of the computational tools used to assesspharmacologic synergy. FIG. 2, Panal A, shows the Combination Index (CI)calculation and FIG. 2, Panel B, shows an isobologram analysis graph.

FIG. 3 is a graph showing the anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib in the NCI-H522 NSCLC xenograft model.

FIG. 4 is a graph showing the combinatorial anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with sorafenib and sunitinib on various cancer celllines.

FIG. 5 is an illustration showing the combinatorial anti-proliferativeeffect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with sorafenib and sunitinib on various cancer celllines.

FIG. 6 is a graph showing the anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with erlotinib in a NCI-H441 human lung tumor xenograftmodel.

FIG. 7 is a graph showing the anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with gefitinib in a NCI-H441 human lung tumor xenograftmodel.

FIG. 8 is a graph showing the dose response curves for thecombinatinorial treatment of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand gemcitabine in pancreatic cell lines.

FIG. 9 is a graph depicting the volume of a MKN-45 human gastric tumorin a xenograft model as a proportion of its initial volume (V/Vo)following treatment with various doses of vehicle (control),(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Agent A), Docetaxel (DTX), or a combination thereof. # indicates p<0.05vs. DTX treatment alone by a student's t-test. ## indicates p<0.01 vs.DTX treatment alone by a student's t-test.

FIG. 10 is a graph depicting the volume of a Hsc-39 human gastric tumorin a xenograft model as a proportion of its initial volume (V/Vo)following treatment with various doses of vehicle,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Agent A), Docetaxel (DTX), or a combination thereof. # indicates p<0.05vs. DTX treatment alone by a stuendent's t-test. ## indicates p<0.01 vs.DTX treatment alone by a student's t-test.

FIG. 11 is a graph depicting the volume of a MKN-45 human gastric tumorin a xenograft model as a proportion of its initial volume (V/Vo)following treatment with various doses of vehicle,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Agent A), 5-FU, or a combination thereof. # indicates p<0.05 vs. 5-FUtreatment alone. * indicates p<0.05 vs. Agent A treatment alone by astudent's t-test.

FIG. 12 is a graph depicting the volume of a MKN-45 human gastric tumorin a xenograft model as a proportion of its initial volume (V/Vo)following treatment with various doses of vehicle,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Agent A), TS-1, or a combination thereof. # indicates p<0.05 vs. TS-1treatment alone. * indicates p<0.05 vs. Agent A treatment alone by astudent's t-test.

FIG. 13 is a graph depicting the volume of a MKN-45 human gastric tumorin a xenograft model as a proportion of its initial volume (V/Vo)following treatment with various doses of vehicle,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Agent A), Capecitabine, or a combination thereof. # indicates p<0.05vs. Capecitabine treatment alone. * indicates p<0.05 vs. Agent Atreatment alone by a student's t-test.

FIG. 14 is a graph depicting the volume of a MKN-45 human gastric tumorin a xenograft model as a proportion of its initial volume (V/Vo)following treatment with various doses of vehicle,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(Agent A), CDDP, or a combination thereof.

DETAILED DESCRIPTION OF THE INVENTION 1. Methods of Treatment

The present invention provides methods of treating a cell proliferativedisorder, the method comprising administering to a subject in needthereof a therapeutically effective amount of a compound of formula III,IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable saltthereof, or a prodrug or metabolite thereof, with one or morepharmaceutically acceptable carriers or excipients, alone, or incombination with a therapeutically effective amount of a secondanti-proliferative agent, with one or more pharmaceutically acceptablecarriers or excipients, wherein the cell proliferation disorder istreated.

The present invention provides a pharmaceutical composition for treatinga cell proliferative disorder, comprising a combination of (a) atherapeutically effective amount of a compound of formula III, IIIa,IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt thereof, or aprodrug or metabolite thereof, alone, or in combination with (b) atherapeutically effective amount of a second anti-proliferative agent.One or more pharmaceutically acceptable carriers or excipients is (are)optionally included in the composition.

A second anti-proliferative agent is preferably a secondchemotherapeutic agent.

The cell proliferative disorder can be a precancerous condition orcancer. The cell proliferative disorder can be a hematologic tumor ormalignancy, or a solid tumor (or tumors). This method of treating cancerinclude a reduction in tumor size. Alternatively, or in addition, thecancer is metastatic cancer and this method of treatment includesinhibition of metastatic cancer cell invasion.

The second chemotherapeutic agent (also referred to as ananti-neoplastic agent or anti-proliferative agent) can be an alkylatingagent; an antibiotic; an anti-metabolite; a detoxifying agent; aninterferon; a polyclonal or monoclonal antibody; an EGFR inhibitor; aHER2 inhibitor; a histone deacetylase inhibitor; a hormone; a mitoticinhibitor; an MTOR inhibitor; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitors; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase inhibitor), a cytidine analogue drug or anychemotherapeutic, anti-neoplastic or anti-proliferative agent listed inwww.cancer.org/docroot/cdg/cdg_(—)0.asp.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary antibiotics include, but are not limited to, doxorubicin(Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone);bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal(DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin(Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin(Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or mesna (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodine, tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (In-111 Zevalin); gemtuzumab (Mylotarg);eculizumab (Soliris); ibritumomab (Y-90 Zevalin); denosumab oribritumomab (Zevalin).

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva);panitumumab (Vectibix); PKI-166; canertinib (CI-1033); matuzumab(Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Histone Deacetylase Inhibitors include, but are not limited to,vorinostat (Zolinza).

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron);abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary mTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; orAP23573.

Exemplary kinase inhibitors include, but are not limited to, Bevacizumab(targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux(targets Erb1), Imatinib/Gleevic (targets Bcr-Abl, PDGFRs and c-Kit),Trastuzumab (targets Erb2), Gefitinib/Iressa (targets EGFR), Ranibizumab(targets VEGF), Pegaptanib (targets VEGF), Erlotinib/Tarceva (targetsErb1), Nilotinib (targets Bcr-Abl), Lapatinib (targets Erb1 andErb2/Her2), GW-572016/lapatinib ditosylate (targets HER2/Erb2),Panitumumab/Vectibix (targets EGFR), Vandetinib (targets RET/VEGFR),E7080 (multiple targets including RET and VEGFR), Herceptin (targetsHER2/Erb2), PKI-166 (targets EGFR), Canertinib/CI-1033 (targets EGFR),Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200(targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR),PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targetsVEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targetsFLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targetsSRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targetsJAK), AG-490 (targets JAK), WH1-P154 (targets JAK), WH1-P131 (targetsJAK), sorafenib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-β, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/Abl and Src),AC-220 (targets Flt3), AC-480 (targets all HER proteins, “pan-HER”),Motesanib diphosphate (targets VEGF1-3, PDGFR, and c-kit), Denosumab(targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534(multiple targets including Flt3).

Exemplary multi-kinase inhibitors include, but are not limited to,sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474;PKC-412; motesanib; or AP24534.

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, eril/easudil hydrochloride; Rapamune (targets mTOR/FRAP1);Deforolimus (targets mTOR); Certican/Everolimus (targets mTOR/FRAP1);AP23573 (targets mTOR/FRAP1); Eril/Fasudil hydrochloride (targets RHO);Flavopiridol (targets CDK); Seliciclib/CYC202/Roscovitrine (targetsCDKs); SNS-032/BMS-387032 (targets CDKs); Ruboxistaurin (targets PKC);Pkc412 (targets PKC); Bryostatin (targets PKC); KAI-9803 (targets PKC);SF1126 (targets PI3K); VX-680 (targets Aurora kinase); Azd1152 (targetsAurora kinase); Arry-142886/AZD-6244 (targets MAP/MEK); SCIO-469(targets MAP/MEK); GW681323 (targets MAP/MEK); CC-401 (targets JNK);CEP-1347 (targets JNK); and PD 332991 (targets CDKs).

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient);dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584);CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606CP-690; AG-490; WH1-P154; WH1-P131; AC-220; or AMG888.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary aromatase inhibitors include, but are not limited to,aminoglutethimide, testolactone (Teslac), anastrozole (Arimidex),Letrozole (Femara), exemestane (Aromasin), Vorozole (Rivizor),Formestane (Lentaron), Fadrozole (Afema), 4-androstene-3,6,17-trione(6-OXO), 1,4,6-androstatrien-3,17-dione (ATD), and4-hydroxyandrostenedione.

Exemplary anthracyclines include, but are not limited to, daunorubicin(Daunomycin), doxorubicin (Adriamycin), epirubicin, idarubicin, andvalrubicin.

Exemplary cytidine analogs include, but are not limited to, gemcitabine,azacytidine (e.g., 5-azacytidine), and cytosine arabinoside (cytarabin,araC, Cytosar).

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxel.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexylen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak);porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid);bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel);arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine(Leucenol); (1M tegafur-0.4 M 5-chloro-2,4-dihydroxypyrimidine-1 Mpotassium oxonate) or statins (e.g., lovastatin, atorvastatin,cerivastatin, fluvastatin, mevastatin, pitavastatin, pravastatin,rosuvastatin, and simvastatin).

In another aspect, the second chemotherapeutic agent can be a cytokinesuch as G-CSF (granulocyte colony stimulating factor). In anotheraspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, may be administered in combination with radiationtherapy. Radiation therapy can also be administered in combination witha compound of formula III, IIIa, IVa, IVb, Va, or Vb and anotherchemotherapeutic agent described herein as part of a multiple agenttherapy. In yet another aspect, a compound of formula III, IIIa, IVa,IVb, Va, or Vb, or a pharmaceutically acceptable salt, prodrug,metabolite, analog or derivative thereof, may be administered incombination with standard chemotherapy combinations such as, but notrestricted to, CMF (cyclophosphamide, methotrexate and 5-fluorouracil),CAF (cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycinand cyclophosphamide), FEC (5-fluorouracil, epirubicin, andcyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, andpaclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP),Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molarratio of 1:0.4:1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar™) orCMFP (cyclophosphamide, methotrexate, 5-fluorouracil and prednisone).

In preferred embodiments, a compound of formula III, IIIa, IVa, IVb, Va,or Vb, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, may be administered with an inhibitor ofan enzyme, such as a receptor or non-receptor kinase. Receptor andnon-receptor kinases of the invention are, for example, tyrosine kinasesor serine/threonine kinases. Kinase inhibitors of the invention aresmall molecules, polynucleic acids, polypeptides, or antibodies.

Preferred combinatorial therapies include, but are not limited to,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneadministered in combination with erlotinib,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneadministered in combination with sorafenib,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneadministered in combination with sunitinib;(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneadministered in combination with capecitabine;(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneadministered in combination with carboplatin and(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneadministered in combination with cisplatin. In certain embodiments, asubject or patient receives a combination of erlotinib, administered as150 mg once daily, in combination with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,administered as 360 mg twice daily. In another embodiment, a subject orpatient receives a combination of sorafenib, administered as 200 mgtwice daily, in combination with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,administered as 360 mg twice daily. Preferred dosage forms for(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinclude, but are not limited to, a capsule and a tablet.

The present examples demonstrate at least an additive anti-proliferativeeffect for various cancers, in vitro and in vivo, when(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered in combination with sorafenib, sunitinib, erlotinib,gefitinib, cisplatin, carboplatin or capecitabine. These cancersinclude, but are not limited to, lung cancer, small cell lung cancer,non-small cell lung cancer, colon cancer, breast cancer, pancreaticcancer, prostate cancer, renal cancer, cervical cancer, brain cancer,gastric/stomach cancer, uterine cancer, intestinal cancer, hepaticcancer, chronic myelogenous leukemia, melanoma, ovarian cancer,translocation-associated renal cell carcinoma (RCC), alveolar soft partsarcoma (ASPS), clear cell sarcoma (CCS), and hepatocellular carcinoma.The anti-proliferative effects of these combinational treatments areincreased/enhanced in cell proliferative disorders and cancers in whichthe effected cells constitutively express or over-express c-Met. Inaddition, synergistic anti-proliferative effects are shown by(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with sorafenib in breast cancer, cervical cancer, lungcancer, small cell lung cancer, non-small cell lung cancer, melanoma,colon cancer, pancreatic cancer, renal cancer and gastric/stomachcancer; by(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with sunitinib in gastric/stomach cancer; by(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with erlotinib in lung cancer, small cell lung cancer,non-small cell lung cancer and colon cancer; and by(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionein combination with cisplatin in pancreatic cancer.

A compound of formula III, IIIa, IVa, IVb, Va, or Vb of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof, can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the compound (i.e. including the active compound), and apharmaceutically acceptable excipient or carrier. As used herein,“pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Suitable carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, a standard referencetext in the field. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin.

Pharmaceutically acceptable carriers include solid carriers such aslactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary liquid carriersinclude syrup, peanut oil, olive oil, water and the like. Similarly, thecarrier or diluent may include time-delay material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or with awax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate orthe like. Other fillers, excipients, flavorants, and other additivessuch as are known in the art may also be included in a pharmaceuticalcomposition according to this invention. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In one aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb, ora pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, is administered in a suitable dosage form preparedby combining a therapeutically effective amount (e.g., an efficaciouslevel sufficient to achieve the desired therapeutic effect throughinhibition of tumor growth, killing of tumor cells, treatment orprevention of cell proliferative disorders, etc.) of a compound offormula III, Ma, IVa, IVb, Va, or Vb, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof, (as an activeingredient) with standard pharmaceutical carriers or diluents accordingto conventional procedures (i.e., by producing a pharmaceuticalcomposition of the invention). These procedures may involve mixing,granulating, and compressing or dissolving the ingredients asappropriate to attain the desired preparation.

As used herein, a “subject” can be any mammal, e.g., a human, a primate,mouse, rat, dog, cat, cow, horse, pig, sheep, goat, camel. In apreferred aspect, the subject is a human.

As used herein, a “subject in need thereof” is a subject having a cellproliferative disorder, or a subject having an increased risk ofdeveloping a cell proliferative disorder relative to the population atlarge. In one aspect, a subject in need thereof has a precancerouscondition. In a preferred aspect, a subject in need thereof has cancer.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue. A cell proliferative disorder includes aprecancer or a precancerous condition. A cell proliferative disorderincludes cancer. Preferably, the methods provided herein are used totreat or alleviate a symptom of cancer. The term “cancer” includes solidtumors, as well as, hematologic tumors and/or malignancies. A “precancercell” or “precancerous cell” is a cell manifesting a cell proliferativedisorder that is a precancer or a precancerous condition. A “cancercell” or “cancerous cell” is a cell manifesting a cell proliferativedisorder that is a cancer. Any reproducible means of measurement may beused to identify cancer cells or precancerous cells. Cancer cells orprecancerous cells can be identified by histological typing or gradingof a tissue sample (e.g., a biopsy sample). Cancer cells or precancerouscells can be identified through the use of appropriate molecularmarkers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, uringarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodeimaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi's sarcoma, kidney cancer, renal cancer, laryngealcancer, acute lymphoblastic leukemia, acute myeloid leukemia, chroniclymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia,lip and oral cavity cancer, liver cancer, lung cancer, non-small celllung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkinlymphoma, primary central nervous system lymphoma, Waldenstrammacroglobulinemia, medulloblastoma, melanoma, intraocular (eye)melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma,metastatic squamous neck cancer, mouth cancer, cancer of the tongue,multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, uterine cancer, uterinesarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel cellskin carcinoma, small intestine cancer, soft tissue sarcoma, squamouscell carcinoma, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, testicular cancer, throat cancer, thymoma,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancerof the renal pelvis and ureter and other urinary organs, gestationaltrophoblastic tumor, urethral cancer, endometrial uterine cancer,uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer,and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. In oneaspect, a cell proliferative disorder of the hematologic system includeslymphoma, leukemia, myeloid neoplasms, mast cell neoplasms,myelodysplasia, benign monoclonal gammopathy, lymphomatoidgranulomatosis, lymphomatoid papulosis, polycythemia vera, chronicmyelocytic leukemia, agnogenic myeloid metaplasia, and essentialthrombocythemia. In another aspect, a cell proliferative disorder of thehematologic system includes hyperplasia, dysplasia, and metaplasia ofcells of the hematologic system. In a preferred aspect, compositions ofthe present invention may be used to treat a cancer selected from thegroup consisting of a hematologic cancer of the present invention or ahematologic cell proliferative disorder of the present invention. In oneaspect, a hematologic cancer of the present invention includes multiplemyeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma,childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin),leukemia (including childhood leukemia, hairy-cell leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, chronic lymphocyticleukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, andmast cell leukemia), myeloid neoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. In one aspect, cell proliferativedisorders of the lung include all forms of cell proliferative disordersaffecting lung cells. In one aspect, cell proliferative disorders of thelung include lung cancer, a precancer or precancerous condition of thelung, benign growths or lesions of the lung, and malignant growths orlesions of the lung, and metastatic lesions in tissue and organs in thebody other than the lung. In a preferred aspect, compositions of thepresent invention may be used to treat lung cancer or cell proliferativedisorders of the lung. In one aspect, lung cancer includes all forms ofcancer of the lung. In another aspect, lung cancer includes malignantlung neoplasms, carcinoma in situ, typical carcinoid tumors, andatypical carcinoid tumors. In another aspect, lung cancer includes smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Inanother aspect, lung cancer includes “scar carcinoma,” bronchioalveolarcarcinoma, giant cell carcinoma, spindle cell carcinoma, and large cellneuroendocrine carcinoma. In another aspect, lung cancer includes lungneoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

In one aspect, cell proliferative disorders of the lung include allforms of cell proliferative disorders affecting lung cells. In oneaspect, cell proliferative disorders of the lung include lung cancer,precancerous conditions of the lung. In one aspect, cell proliferativedisorders of the lung include hyperplasia, metaplasia, and dysplasia ofthe lung. In another aspect, cell proliferative disorders of the lunginclude asbestos-induced hyperplasia, squamous metaplasia, and benignreactive mesothelial metaplasia. In another aspect, cell proliferativedisorders of the lung include replacement of columnar epithelium withstratified squamous epithelium, and mucosal dysplasia. In anotheraspect, individuals exposed to inhaled injurious environmental agentssuch as cigarette smoke and asbestos may be at increased risk fordeveloping cell proliferative disorders of the lung. In another aspect,prior lung diseases that may predispose individuals to development ofcell proliferative disorders of the lung include chronic interstitiallung disease, necrotizing pulmonary disease, scleroderma, rheumatoiddisease, sarcoidosis, interstitial pneumonitis, tuberculosis, repeatedpneumonias, idiopathic pulmonary fibrosis, granulomata, asbestosis,fibrosing alveolitis, and Hodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. In a preferred aspect, the cellproliferative disorder of the colon is colon cancer. In a preferredaspect, compositions of the present invention may be used to treat coloncancer or cell proliferative disorders of the colon. In one aspect,colon cancer includes all forms of cancer of the colon. In anotheraspect, colon cancer includes sporadic and hereditary colon cancers. Inanother aspect, colon cancer includes malignant colon neoplasms,carcinoma in situ, typical carcinoid tumors, and atypical carcinoidtumors. In another aspect, colon cancer includes adenocarcinoma,squamous cell carcinoma, and adenosquamous cell carcinoma. In anotheraspect, colon cancer is associated with a hereditary syndrome selectedfrom the group consisting of hereditary nonpolyposis colorectal cancer,familial adenomatous polyposis, Gardner's syndrome, Peutz-Jegherssyndrome, Turcot's syndrome and juvenile polyposis. In another aspect,colon cancer is caused by a hereditary syndrome selected from the groupconsisting of hereditary nonpolyposis colorectal cancer, familialadenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome,Turcot's syndrome and juvenile polyposis.

In one aspect, cell proliferative disorders of the colon include allforms of cell proliferative disorders affecting colon cells. In oneaspect, cell proliferative disorders of the colon include colon cancer,precancerous conditions of the colon, adenomatous polyps of the colonand metachronous lesions of the colon. In one aspect, a cellproliferative disorder of the colon includes adenoma. In one aspect,cell proliferative disorders of the colon are characterized byhyperplasia, metaplasia, and dysplasia of the colon. In another aspect,prior colon diseases that may predispose individuals to development ofcell proliferative disorders of the colon include prior colon cancer. Inanother aspect, current disease that may predispose individuals todevelopment of cell proliferative disorders of the colon include Crohn'sdisease and ulcerative colitis. In one aspect, a cell proliferativedisorder of the colon is associated with a mutation in a gene selectedfrom the group consisting of p53, ras, FAP and DCC. In another aspect,an individual has an elevated risk of developing a cell proliferativedisorder of the colon due to the presence of a mutation in a geneselected from the group consisting of p53, ras, FAP and DCC.

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. In one aspect, cellproliferative disorders of the prostate include all forms of cellproliferative disorders affecting prostate cells. In one aspect, cellproliferative disorders of the prostate include prostate cancer, aprecancer or precancerous condition of the prostate, benign growths orlesions of the prostate, and malignant growths or lesions of theprostate, and metastatic lesions in tissue and organs in the body otherthan the prostate. In another aspect, cell proliferative disorders ofthe prostate include hyperplasia, metaplasia, and dysplasia of theprostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. In one aspect, cell proliferativedisorders of the skin include all forms of cell proliferative disordersaffecting skin cells. In one aspect, cell proliferative disorders of theskin include a precancer or precancerous condition of the skin, benigngrowths or lesions of the skin, melanoma, malignant melanoma and othermalignant growths or lesions of the skin, and metastatic lesions intissue and organs in the body other than the skin. In another aspect,cell proliferative disorders of the skin include hyperplasia,metaplasia, psoriasis, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. In one aspect, cell proliferativedisorders of the ovary include all forms of cell proliferative disordersaffecting cells of the ovary. In one aspect, cell proliferativedisorders of the ovary include a precancer or precancerous condition ofthe ovary, benign growths or lesions of the ovary, ovarian cancer,malignant growths or lesions of the ovary, and metastatic lesions intissue and organs in the body other than the ovary. In another aspect,cell proliferative disorders of the ovary include hyperplasia,metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. In one aspect, cellproliferative disorders of the breast include all forms of cellproliferative disorders affecting breast cells. In one aspect, cellproliferative disorders of the breast include breast cancer, a precanceror precancerous condition of the breast, benign growths or lesions ofthe breast, and malignant growths or lesions of the breast, andmetastatic lesions in tissue and organs in the body other than thebreast. In another aspect, cell proliferative disorders of the breastinclude hyperplasia, metaplasia, and dysplasia of the breast.

In one aspect, a cell proliferative disorder of the breast is aprecancerous condition of the breast. In one aspect, compositions of thepresent invention may be used to treat a precancerous condition of thebreast. In one aspect, a precancerous condition of the breast includesatypical hyperplasia of the breast, ductal carcinoma in situ (DCIS),intraductal carcinoma, lobular carcinoma in situ (LCIS), lobularneoplasia, and stage 0 or grade 0 growth or lesion of the breast (e.g.,stage 0 or grade 0 breast cancer, or carcinoma in situ). In anotheraspect, a precancerous condition of the breast has been staged accordingto the TNM classification scheme as accepted by the American JointCommittee on Cancer (AJCC), where the primary tumor (T) has beenassigned a stage of T0 or T is; and where the regional lymph nodes (N)have been assigned a stage of NO; and where distant metastasis (M) hasbeen assigned a stage of MO.

In a preferred aspect, the cell proliferative disorder of the breast isbreast cancer. In a preferred aspect, compositions of the presentinvention may be used to treat breast cancer. In one aspect, breastcancer includes all forms of cancer of the breast. In one aspect, breastcancer includes primary epithelial breast cancers. In another aspect,breast cancer includes cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. In another aspect, breastcancer includes carcinoma of the breast, ductal carcinoma of the breast,lobular carcinoma of the breast, undifferentiated carcinoma of thebreast, cystosarcoma phyllodes of the breast, angiosarcoma of thebreast, and primary lymphoma of the breast. In one aspect, breast cancerincludes Stage I, II, IIIA, IIIB, IIIC and IV breast cancer. In oneaspect, ductal carcinoma of the breast includes invasive carcinoma,invasive carcinoma in situ with predominant intraductal component,inflammatory breast cancer, and a ductal carcinoma of the breast with ahistologic type selected from the group consisting of comedo, mucinous(colloid), medullary, medullary with lymphcytic infiltrate, papillary,scirrhous, and tubular. In one aspect, lobular carcinoma of the breastincludes invasive lobular carcinoma with predominant in situ component,invasive lobular carcinoma, and infiltrating lobular carcinoma. In oneaspect, breast cancer includes Paget's disease, Paget's disease withintraductal carcinoma, and Paget's disease with invasive ductalcarcinoma. In another aspect, breast cancer includes breast neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

In a preferred aspect, a compound of formula III, IIIa, IVa, IVb, Va, orVb may be used to treat breast cancer. In one aspect, a breast cancerthat is to be treated includes familial breast cancer. In anotheraspect, a breast cancer that is to be treated includes sporadic breastcancer. In one aspect, a breast cancer that is to be treated has arisenin a male subject. In one aspect, a breast cancer that is to be treatedhas arisen in a female subject. In one aspect, a breast cancer that isto be treated has arisen in a premenopausal female subject or apostmenopausal female subject. In one aspect, a breast cancer that is tobe treated has arisen in a subject equal to or older than 30 years old,or a subject younger than 30 years old. In one aspect, a breast cancerthat is to be treated has arisen in a subject equal to or older than 50years old, or a subject younger than 50 years old. In one aspect, abreast cancer that is to be treated has arisen in a subject equal to orolder than 70 years old, or a subject younger than 70 years old.

In one aspect, a breast cancer that is to be treated has been typed toidentify a familial or spontaneous mutation in BRCA1, BRCA2, or p53. Inone aspect, a breast cancer that is to be treated has been typed ashaving a HER2/neu gene amplification, as overexpressing HER2/neu, or ashaving a low, intermediate or high level of HER2/neu expression. Inanother aspect, a breast cancer that is to be treated has been typed fora marker selected from the group consisting of estrogen receptor (ER),progesterone receptor (PR), human epidermal growth factor receptor-2,Ki-67, CA15-3, CA 27-29, and c-Met. In one aspect, a breast cancer thatis to be treated has been typed as ER-unknown, ER-rich or ER-poor. Inanother aspect, a breast cancer that is to be treated has been typed asER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. In a preferred aspect, ER-typing ofa breast cancer may be performed as set forth in Onkologie 27: 175-179(2004). In one aspect, a breast cancer that is to be treated has beentyped as PR-unknown, PR-rich or PR-poor. In another aspect, a breastcancer that is to be treated has been typed as PR-negative orPR-positive. In another aspect, a breast cancer that is to be treatedhas been typed as receptor positive or receptor negative. In one aspect,a breast cancer that is to be treated has been typed as being associatedwith elevated blood levels of CA 15-3, or CA 27-29, or both.

In one aspect, a breast cancer that is to be treated includes alocalized tumor of the breast. In one aspect, a breast cancer that is tobe treated includes a tumor of the breast that is associated with anegative sentinel lymph node (SLN) biopsy. In one aspect, a breastcancer that is to be treated includes a tumor of the breast that isassociated with a positive sentinel lymph node (SLN) biopsy. In anotheraspect, a breast cancer that is to be treated includes a tumor of thebreast that is associated with one or more positive auxiliary lymphnodes, where the auxiliary lymph nodes have been staged by anyapplicable method. In another aspect, a breast cancer that is to betreated includes a tumor of the breast that has been typed as havingnodal negative status (e.g., node-negative) or nodal positive status(e.g., node-positive). In another aspect, a breast cancer that is to betreated includes a tumor of the breast that has metastasized to otherlocations in the body. In one aspect, a breast cancer that is to betreated is classified as having metastasized to a location selected fromthe group consisting of bone, lung, liver, or brain. In another aspect abreast cancer that is to be treated is classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

In one aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb maybe used to treat or prevent a cell proliferative disorder of the breast,or to treat or prevent breast cancer, in a subject having an increasedrisk of developing breast cancer relative to the population at large. Inone aspect, a subject with an increased risk of developing breast cancerrelative to the population at large is a female subject with a familyhistory or personal history of breast cancer. In another aspect, asubject with an increased risk of developing breast cancer relative tothe population at large is a female subject having a germ-line orspontaneous mutation in BRCA1 or BRCA2, or both. In one aspect, asubject with an increased risk of developing breast cancer relative tothe population at large is a female subject with a family history ofbreast cancer and a germ-line or spontaneous mutation in BRCA1 or BRCA2,or both. In another aspect, a subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalewho is greater than 30 years old, greater than 40 years old, greaterthan 50 years old, greater than 60 years old, greater than 70 years old,greater than 80 years old, or greater than 90 years old. In one aspect,a subject with an increased risk of developing breast cancer relative tothe population at large is a subject with atypical hyperplasia of thebreast, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, or a stage 0 growth orlesion of the breast (e.g., stage 0 or grade 0 breast cancer, orcarcinoma in situ).

In another aspect, a breast cancer that is to be treated has beenhistologically graded according to the Scarff-Bloom-Richardson system,wherein a breast tumor has been assigned a mitosis count score of 1, 2,or 3; a nuclear pleiomorphism score of 1, 2, or 3; a tubule formationscore of 1, 2, or 3; and a total Scarff-Bloom-Richardson score ofbetween 3 and 9. In another aspect, a breast cancer that is to betreated has been assigned a tumor grade according to the InternationalConsensus Panel on the Treatment of Breast Cancer selected from thegroup consisting of grade 1, grade 1-2, grade 2, grade 2-3, or grade 3.

In one aspect, a cancer that is to be treated has been staged accordingto the American Joint Committee on Cancer (AJCC) TNM classificationsystem, where the tumor (T) has been assigned a stage of TX, T1, T1mic,T1a, T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and where the regionallymph nodes (N) have been assigned a stage of NX, N0, N1, N2, N2a, N2b,N3, N3a, N3b, or N3c; and where distant metastasis (M) has been assigneda stage of MX, MO, or Ml. In another aspect, a cancer that is to betreated has been staged according to an American Joint Committee onCancer (AJCC) classification as Stage I, Stage IIA, Stage IIB, StageIIIA, Stage IIIB, Stage IIIC, or Stage IV. In another aspect, a cancerthat is to be treated has been assigned a grade according to an AJCCclassification as Grade GX (e.g., grade cannot be assessed), Grade 1,Grade 2, Grade 3 or Grade 4. In another aspect, a cancer that is to betreated has been staged according to an AJCC pathologic classification(pN) of pNX, pN0, PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1,PN1(mi), PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

In one aspect, a cancer that is to be treated includes a tumor that hasbeen determined to be less than or equal to about 2 centimeters indiameter. In another aspect, a cancer that is to be treated includes atumor that has been determined to be from about 2 to about 5 centimetersin diameter. In another aspect, a cancer that is to be treated includesa tumor that has been determined to be greater than or equal to about 3centimeters in diameter. In another aspect, a cancer that is to betreated includes a tumor that has been determined to be greater than 5centimeters in diameter. In another aspect, a cancer that is to betreated is classified by microscopic appearance as well differentiated,moderately differentiated, poorly differentiated, or undifferentiated.In another aspect, a cancer that is to be treated is classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Inanother aspect, a cancer that is to be treated is classified bymicroscopic appearance as being associated with areas of necrosis (e.g.,areas of dying or degenerating cells). In one aspect, a cancer that isto be treated is classified as having an abnormal karyotype, having anabnormal number of chromosomes, or having one or more chromosomes thatare abnormal in appearance. In one aspect, a cancer that is to betreated is classified as being aneuploid, triploid, tetraploid, or ashaving an altered ploidy. In one aspect, a cancer that is to be treatedis classified as having a chromosomal translocation, or a deletion orduplication of an entire chromosome, or a region of deletion,duplication or amplification of a portion of a chromosome.

In one aspect, a cancer that is to be treated is evaluated by DNAcytometry, flow cytometry, or image cytometry. In one aspect, a cancerthat is to be treated has been typed as having 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% of cells in the synthesis stage of cell division(e.g., in S phase of cell division). In one aspect, a cancer that is tobe treated has been typed as having a low S-phase fraction or a highS-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder.” In one aspect, a normal celllacks unregulated or abnormal growth, or both, that can lead to thedevelopment of an unwanted condition or disease. Preferably, a normalcell possesses normally functioning cell cycle checkpoint controlmechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of formulaIII, IIIa, IVa, IVb, Va, or Vb that has been or will be tested in one ormore in vitro or in vivo biological assays, in order to determine ifthat compound is likely to elicit a desired biological or medicalresponse in a cell, tissue, system, animal or human that is being soughtby a researcher or clinician. In one aspect, a candidate compound is acompound of formula III or IIIa; in another aspect, a candidate compoundis a compound of formula IVa, IVb, Va, or Vb. In a preferred aspect, thebiological or medical response is treatment of cancer. In anotheraspect, the biological or medical response is treatment or prevention ofa cell proliferative disorder. In one aspect, in vitro or in vivobiological assays include, but are not limited to, enzymatic activityassays, electrophoretic mobility shift assays, reporter gene assays, invitro cell viability assays, and the assays.

As used herein, “monotherapy” refers to administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an active compound. For example, cancer monotherapy with(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecomprises administration of a therapeutically effective amount of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, to a subject in need of treatment of cancer.Monotherapy may be contrasted with combination therapy, in which acombination of multiple active compounds is administered, preferablywith each component of the combination present in a therapeuticallyeffective amount. In one aspect, montherapy with a compound of formulaIII, IIIa, IVa, IVb, Va, or Vb is more effective than combinationtherapy in inducing a desired biological effect. Monotherapeuticeffectiveness of the compounds of formula III, IIIa, IVa, IVb, Va, or Vbis shown in PCT Publication No. WO 2006/086484.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes decreasing or alleviating the symptoms or complications, oreliminating the disease, condition or disorder.

As used herein, “preventing” describes stopping the onset of thesymptoms or complications of the disease, condition or disorder.

In one aspect, treating cancer results in a reduction in size of atumor. A reduction in size of a tumor may also be referred to as “tumorregression.” Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. In a preferred aspect, size of a tumor may be measured as adiameter of the tumor.

In another aspect, treating cancer results in a reduction in tumorvolume. Preferably, after treatment, tumor volume is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorvolume is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Tumor volume may be measured by any reproducible means of measurement.

In another aspect, treating cancer results in a decrease in number oftumors. Preferably, after treatment, tumor number is reduced by 5% orgreater relative to number prior to treatment; more preferably, tumornumber is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75%. Number oftumors may be measured by any reproducible means of measurement. In apreferred aspect, number of tumors may be measured by counting tumorsvisible to the naked eye or at a specified magnification. In a preferredaspect, the specified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

In another aspect, treating cancer results in a decrease in number ofmetastatic lesions in other tissues or organs distant from the primarytumor site. Preferably, after treatment, the number of metastaticlesions is reduced by 5% or greater relative to number prior totreatment; more preferably, the number of metastatic lesions is reducedby 10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. The number of metastaticlesions may be measured by any reproducible means of measurement. In apreferred aspect, the number of metastatic lesions may be measured bycounting metastatic lesions visible to the naked eye or at a specifiedmagnification. In a preferred aspect, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

In another aspect, treating cancer results in an increase in averagesurvival time of a population of treated subjects in comparison to apopulation receiving carrier alone. Preferably, the average survivaltime is increased by more than 30 days; more preferably, by more than 60days; more preferably, by more than 90 days; and most preferably, bymore than 120 days. An increase in average survival time of a populationmay be measured by any reproducible means. In a preferred aspect, anincrease in average survival time of a population may be measured, forexample, by calculating for a population the average length of survivalfollowing initiation of treatment with an active compound. In anotherpreferred aspect, an increase in average survival time of a populationmay also be measured, for example, by calculating for a population theaverage length of survival following completion of a first round oftreatment with an active compound.

In another aspect, treating cancer results in an increase in averagesurvival time of a population of treated subjects in comparison to apopulation of untreated subjects. Preferably, the average survival timeis increased by more than 30 days; more preferably, by more than 60days; more preferably, by more than 90 days; and most preferably, bymore than 120 days. An increase in average survival time of a populationmay be measured by any reproducible means. In a preferred aspect, anincrease in average survival time of a population may be measured, forexample, by calculating for a population the average length of survivalfollowing initiation of treatment with an active compound. In anotherpreferred aspect, an increase in average survival time of a populationmay also be measured, for example, by calculating for a population theaverage length of survival following completion of a first round oftreatment with an active compound.

In another aspect, treating cancer results in increase in averagesurvival time of a population of treated subjects in comparison to apopulation receiving monotherapy with a drug that is not a compound offormula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof. Preferably, theaverage survival time is increased by more than 30 days; morepreferably, by more than 60 days; more preferably, by more than 90 days;and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.In a preferred aspect, an increase in average survival time of apopulation may be measured, for example, by calculating for a populationthe average length of survival following initiation of treatment with anactive compound. In another preferred aspect, an increase in averagesurvival time of a population may also be measured, for example, bycalculating for a population the average length of survival followingcompletion of a first round of treatment with an active compound.

In another aspect, treating cancer results in a decrease in themortality rate of a population of treated subjects in comparison to apopulation receiving carrier alone. In another aspect, treating cancerresults in a decrease in the mortality rate of a population of treatedsubjects in comparison to an untreated population. In a further aspect,treating cancer results a decrease in the mortality rate of a populationof treated subjects in comparison to a population receiving monotherapywith a drug that is not a compound of formula III, IIIa, IVa, IVb, Va,or Vb, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Ina preferred aspect, a decrease in the mortality rate of a population oftreated subjects may be measured by any reproducible means. In anotherpreferred aspect, a decrease in the mortality rate of a population maybe measured, for example, by calculating for a population the averagenumber of disease-related deaths per unit time following initiation oftreatment with an active compound. In another preferred aspect, adecrease in the mortality rate of a population may also be measured, forexample, by calculating for a population the average number ofdisease-related deaths per unit time following completion of a firstround of treatment with an active compound.

In another aspect, treating cancer results in a decrease in tumor growthrate. Preferably, after treatment, tumor growth rate is reduced by atleast 5% relative to number prior to treatment; more preferably, tumorgrowth rate is reduced by at least 10%; more preferably, reduced by atleast 20%; more preferably, reduced by at least 30%; more preferably,reduced by at least 40%; more preferably, reduced by at least 50%; evenmore preferably, reduced by at least 50%; and most preferably, reducedby at least 75%. Tumor growth rate may be measured by any reproduciblemeans of measurement. In a preferred aspect, tumor growth rate ismeasured according to a change in tumor diameter per unit time.

In another aspect, treating cancer results in a decrease in tumorregrowth. Preferably, after treatment, tumor regrowth is less than 5%;more preferably, tumor regrowth is less than 10%; more preferably, lessthan 20%; more preferably, less than 30%; more preferably, less than40%; more preferably, less than 50%; even more preferably, less than50%; and most preferably, less than 75%. Tumor regrowth may be measuredby any reproducible means of measurement. In a preferred aspect, tumorregrowth is measured, for example, by measuring an increase in thediameter of a tumor after a prior tumor shrinkage that followedtreatment. In another preferred aspect, a decrease in tumor regrowth isindicated by failure of tumors to reoccur after treatment has stopped.

In another aspect, treating or preventing a cell proliferative disorderresults in a reduction in the rate of cellular proliferation.Preferably, after treatment, the rate of cellular proliferation isreduced by at least 5%; more preferably, by at least 10%; morepreferably, by at least 20%; more preferably, by at least 30%; morepreferably, by at least 40%; more preferably, by at least 50%; even morepreferably, by at least 50%; and most preferably, by at least 75%. Therate of cellular proliferation may be measured by any reproducible meansof measurement. In a preferred aspect, the rate of cellularproliferation is measured, for example, by measuring the number ofdividing cells in a tissue sample per unit time.

In another aspect, treating or preventing a cell proliferative disorderresults in a reduction in the proportion of proliferating cells.Preferably, after treatment, the proportion of proliferating cells isreduced by at least 5%; more preferably, by at least 10%; morepreferably, by at least 20%; more preferably, by at least 30%; morepreferably, by at least 40%; more preferably, by at least 50%; even morepreferably, by at least 50%; and most preferably, by at least 75%. Theproportion of proliferating cells may be measured by any reproduciblemeans of measurement. In a preferred aspect, the proportion ofproliferating cells is measured, for example, by quantifying the numberof dividing cells relative to the number of nondividing cells in atissue sample. In another preferred aspect, the proportion ofproliferating cells is equivalent to the mitotic index.

In another aspect, treating or preventing a cell proliferative disorderresults in a decrease in size of an area or zone of cellularproliferation. Preferably, after treatment, size of an area or zone ofcellular proliferation is reduced by at least 5% relative to its sizeprior to treatment; more preferably, reduced by at least 10%; morepreferably, reduced by at least 20%; more preferably, reduced by atleast 30%; more preferably, reduced by at least 40%; more preferably,reduced by at least 50%; even more preferably, reduced by at least 50%;and most preferably, reduced by at least 75%. Size of an area or zone ofcellular proliferation may be measured by any reproducible means ofmeasurement. In a preferred aspect, size of an area or zone of cellularproliferation may be measured as a diameter or width of an area or zoneof cellular proliferation.

In another aspect, treating or preventing a cell proliferative disorderresults in a decrease in the number or proportion of cells having anabnormal appearance or morphology. Preferably, after treatment, thenumber of cells having an abnormal morphology is reduced by at least 5%relative to its size prior to treatment; more preferably, reduced by atleast 10%; more preferably, reduced by at least 20%; more preferably,reduced by at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. In one aspect, an abnormal cellular morphology ismeasured by microscopy, e.g., using an inverted tissue culturemicroscope. In one aspect, an abnormal cellular morphology takes theform of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. In oneaspect, the compared populations are cell populations. In a preferredaspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, acts selectively on a cancer or precancerous cellbut not on a normal cell. In another preferred aspect, a compound offormula III, IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptablesalt, prodrug, metabolite, analog or derivative thereof, actsselectively to modulate one molecular target (e.g., c-Met) but does notsignificantly modulate another molecular target (e.g., Protein KinaseC). In another preferred aspect, the invention provides a method forselectively inhibiting the activity of an enzyme, such as a kinase.Preferably, an event occurs selectively in population A relative topopulation B if it occurs greater than two times more frequently inpopulation A as compared to population B. More preferably, an eventoccurs selectively if it occurs greater than five times more frequentlyin population A. More preferably, an event occurs selectively if itoccurs greater than ten times more frequently in population A; morepreferably, greater than fifty times; even more preferably, greater than100 times; and most preferably, greater than 1000 times more frequentlyin population A as compared to population B. For example, cell deathwould be said to occur selectively in cancer cells if it occurredgreater than twice as frequently in cancer cells as compared to normalcells.

In a preferred aspect, a compound of formula III, IIIa, IVa, IVb, Va, orVb or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, modulates the activity of a molecular target (e.g.,c-Met). In one aspect, modulating refers to stimulating or inhibiting anactivity of a molecular target. Preferably, a compound of formula III,IIIa, IVa, IVb, Va, or Vb modulates the activity of a molecular targetif it stimulates or inhibits the activity of the molecular target by atleast 2-fold relative to the activity of the molecular target under thesame conditions but lacking only the presence of said compound. Morepreferably, a compound of formula III, IIIa, IVa, IVb, Va, or Vbmodulates the activity of a molecular target if it stimulates orinhibits the activity of the molecular target by at least 5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, at least 100-foldrelative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

In one aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vb, ora pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, does not significantly modulate the activity of amolecular target if the addition of the compound does not stimulate orinhibit the activity of the molecular target by greater than 10%relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. In apreferred aspect, a compound of formula III, IIIa, IVa, IVb, Va, or Vbdoes not significantly modulate the activity of Protein Kinase C.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a kinase isozyme alpha in comparison to a kinase isozymebeta). Preferably, a compound of formula III, IIIa, IVa, IVb, Va, or Vbdemonstrates a minimum of a four fold differential, preferably a tenfold differential, more preferably a fifty fold differential, in thedosage required to achieve a biological effect. Preferably, a compoundof formula III, IIIa, IVa, IVb, Va, or Vb demonstrates this differentialacross the range of inhibition, and the differential is exemplified atthe IC₅₀, i.e., a 50% inhibition, for a molecular target of interest.

In a preferred embodiment, administering a compound of formula III,IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,prodrug, metabolite, analog or derivative thereof, to a cell or asubject in need thereof results in modulation (i.e., stimulation orinhibition) of an activity of c-Met. As used herein, an activity ofc-Met refers to any biological function or activity that is carried outby c-Met. For example, a function of c-Met includes phosphorylation ofdownstream target proteins. Other functions of c-Met includeautophosphorylation, binding of adaptor proteins such as Gab-1, Grb-2,Shc, SHP2 and c-Cbl, and activation of signal transducers such as Ras,Src, PI3K, PLC-γ, STATs, ERK1 and 2 and FAK. c-Met knockdown has beenshown to inhibit cancer cell growth in a cell-type-specific manner.MDA-MB-231, NCI-H661, NCI-H441, MIA PaCa-2, HT29 and MKN-45 human cancercells. c-Met knockdown induces caspase-dependent apoptosis in a celltype-specific manner. Thus, the present invention is directed to thetreatment of cell proliferative disorders where the cells express c-Metat high levels or express active c-Met.

In a preferred embodiment, administering a compound of formula III,IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,prodrug, metabolite, analog or derivative thereof, to a cell or asubject in need thereof results in modulation (i.e., stimulation orinhibition) of an activity of ERK 1 or ERK 2, or both. As used herein,an activity of ERK 1 or ERK 2 refers to any biological function oractivity that is carried out by ERK 1 or ERK 2. For example, a functionof ERK 1 or ERK 2 includes phosphorylation of downstream targetproteins.

In one aspect, activating refers to placing a composition of matter(e.g., protein or nucleic acid) in a state suitable for carrying out adesired biological function. In one aspect, a composition of mattercapable of being activated also has an unactivated state. In one aspect,an activated composition of matter may have an inhibitory or stimulatorybiological function, or both.

In one aspect, elevation refers to an increase in a desired biologicalactivity of a composition of matter (e.g., a protein or a nucleic acid).In one aspect, elevation may occur through an increase in concentrationof a composition of matter.

As used herein, “a cell cycle checkpoint pathway” refers to abiochemical pathway that is involved in modulation of a cell cyclecheckpoint. A cell cycle checkpoint pathway may have stimulatory orinhibitory effects, or both, on one or more functions comprising a cellcycle checkpoint. A cell cycle checkpoint pathway is comprised of atleast two compositions of matter, preferably proteins, both of whichcontribute to modulation of a cell cycle checkpoint. A cell cyclecheckpoint pathway may be activated through an activation of one or moremembers of the cell cycle checkpoint pathway. Preferably, a cell cyclecheckpoint pathway is a biochemical signaling pathway.

As used herein, “cell cycle checkpoint regulator” refers to acomposition of matter that can function, at least in part, in modulationof a cell cycle checkpoint. A cell cycle checkpoint regulator may havestimulatory or inhibitory effects, or both, on one or more functionscomprising a cell cycle checkpoint. In one aspect, a cell cyclecheckpoint regulator is a protein. In another aspect, a cell cyclecheckpoint regulator is not a protein.

In one aspect, treating cancer or a cell proliferative disorder resultsin cell death, and preferably, cell death results in a decrease of atleast 10% in number of cells in a population. More preferably, celldeath means a decrease of at least 20%; more preferably, a decrease ofat least 30%; more preferably, a decrease of at least 40%; morepreferably, a decrease of at least 50%; most preferably, a decrease ofat least 75%. Number of cells in a population may be measured by anyreproducible means. In one aspect, number of cells in a population ismeasured by fluorescence activated cell sorting (FACS). In anotheraspect, number of cells in a population is measured byimmunofluorescence microscopy. In another aspect, number of cells in apopulation is measured by light microscopy. In another aspect, methodsof measuring cell death are as shown in Li et al., (2003) Proc Natl AcadSci USA. 100(5): 2674-8. In an aspect, cell death occurs by apoptosis.

In a preferred aspect, an effective amount of a compound of formula III,IIIa, IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt,prodrug, metabolite, analog or derivative thereof is not significantlycytotoxic to normal cells. A therapeutically effective amount of acompound is not significantly cytotoxic to normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. Atherapeutically effective amount of a compound does not significantlyaffect the viability of normal cells if administration of the compoundin a therapeutically effective amount does not induce cell death ingreater than 10% of normal cells. In an aspect, cell death occurs byapoptosis.

In one aspect, contacting a cell with a compound of formula III, IIIa,IVa, IVb, Va, or Vb, or a pharmaceutically acceptable salt, prodrug,metabolite, analog or derivative thereof, induces or activates celldeath selectively in cancer cells. Preferably, administering to asubject in need thereof a compound of formula III, IIIa, IVa, IVb, Va,or Vb, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof, induces or activates cell deathselectively in cancer cells. In another aspect, contacting a cell with acompound of formula III, IIIa, IVa, IVb, Va, or Vb, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, induces cell death selectively in one or more cellsaffected by a cell proliferative disorder. Preferably, administering toa subject in need thereof a compound of formula III, IIIa, IVa, IVb, Va,or Vb, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof, induces cell death selectively in one ormore cells affected by a cell proliferative disorder. In a preferredaspect, the present invention relates to a method of treating orpreventing cancer by administering a compound of formula III, IIIa, IVa,IVb, Va, or Vb, or a pharmaceutically acceptable salt, prodrug,metabolite, analog or derivative thereof to a subject in need thereof,where administration of the compound of formula III, IIIa, IVa, IVb, Va,or Vb, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof results in one or more of the following:accumulation of cells in G1 and/or S phase of the cell cycle,cytotoxicity via cell death in cancer cells without a significant amountof cell death in normal cells, antitumor activity in animals with atherapeutic index of at least 2, and activation of a cell cyclecheckpoint. As used herein, “therapeutic index” is the maximum tolerateddose divided by the efficacious dose. One skilled in the art may referto general reference texts for detailed descriptions of known techniquesdiscussed herein or equivalent techniques. These texts include Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3ded.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2000); Coliganet al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Ennaet al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.;Fingl et al., The Pharmacological Basis of Therapeutics (1975),Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,18th edition (1990). These texts can, of course, also be referred to inmaking or using an aspect of the invention.

2. Pyrroloquinolinyl-pyrrole-2,5-diones andpyrroloquinolinyl-pyrrolidine-2,5-diones

The pyrroloquinolinyl-pyrrole-2,5-dione compounds of formula III andIIIa are:

where:

R1, R2 and R3 are independently selected from the group consisting ofhydrogen, F, Cl, Br, I, —NR5R6, —(C₁-C₆) alkyl, —(C₁-C₆) substitutedalkyl, —(C₃-C₉) cycloalkyl, —(C₃-C₉) substituted cycloalkyl, —O—(C₁-C₆)alkyl, —O—(C₁-C₆) substituted alkyl, —O—(C₃-C₉) cycloalkyl, and—O—(C₃-C₉) substituted cycloalkyl, aryl, heteroaryl, heterocyclyl;

R4 is independently selected from the group consisting of hydrogen,—(C₁-C₆) alkyl, —CH₂R7;

R5, R6 are independently selected from the group consisting of hydrogen,and —(C₁-C₆) alkyl;

R7 is independently selected from the group consisting of —O—P(═O)(OH)₂,—O—P(═O)(—OH)(—O—(C₁-C₆) alkyl), —O—P(═O)(—O—(C₁-C₆) alkyl)₂,—O—P(═O)(—OH) (—O—(CH₂)-phenyl), —O—P(═O)(—O—(CH₂)-phenyl)₂, acarboxylic acid group, an amino carboxylic acid group and a peptide;

Q is selected from the group consisting of aryl, heteroaryl, —O-aryl,—S-aryl, —O-heteroaryl, and —S-heteroaryl;

X is selected from the group consisting of —(CH₂)—, —(NR8)-, S, and O;

R8 is independently selected from the group consisting of hydrogen,—(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉) cycloalkyl,—(C₃-C₉) substituted cycloalkyl, and —O—(C₁-C₆) alkyl, —C(═O)—O—(C₁-C₆)alkyl and —C(═O)—O—(C₁-C₆) substituted alkyl;

Y is selected from the group consisting of —(CH₂)— or a bond;

wherein said aryl, heteroaryl, —O-aryl, —S-aryl, —O-heteroaryl, and—S-heteroaryl groups may be substituted with one or more substituentsindependently selected from the group consisting of F, Cl, Br, I,—NR5R6, —(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉) cycloalkyl,—(C₃-C₉) substituted cycloalkyl, —O—(C₁-C₆) alkyl, —O—(C₁-C₆)substituted alkyl, —O—(C₃-C₉) cycloalkyl, —O—(C₃-C₉) substitutedcycloalkyl, -aryl, -aryl-(C₁-C₆) alkyl, -aryl-O—(C₁-C₆) alkyl, —O-aryl,—O—(C₁-C₄) alkyl-aryl, heteroaryl, heterocyclyl, —O—(C₁-C₄)alkyl-heterocycle, and —(S(═O)₂)—(C₁-C₆) alkyl; and

m is 1 or 2.

For the compound of formula Ma, Q is selected from the group consistingof aryl, heteroaryl, —O-aryl, —S-aryl, —O-heteroaryl, and —S-heteroaryl,provided that when R4 is hydrogen, or (C₁-C₄) alkyl, Q is not 3-indolylor substituted 3-indolyl.

The pyrroloquinolinyl-pyrrolidine-2,5-dione compounds of formula IVa,IVb, Va, or Vb, are:

where:

R1, R2 and R3 are independently selected from the group consisting ofhydrogen, F, Cl, Br, I, —NR5R6, —(C₁-C₆) alkyl, —(C₁-C₆) substitutedalkyl, —(C₃-C₉) cycloalkyl, —(C₃-C₉) substituted cycloalkyl, —O—(C₁-C₆)alkyl, —O—(C₁-C₆) substituted alkyl, —O—(C₃-C₉) cycloalkyl, and—O—(C₃-C₉) substituted cycloalkyl, aryl, heteroaryl, heterocyclyl;

R4 is independently selected from the group consisting of hydrogen,—(C₁-C₆) alkyl, —CH₂R7;

R5, R6 are independently selected from the group consisting of hydrogen,and —(C₁-C₆) alkyl;

R7 is independently selected from the group consisting of —O—P(═O)(OH)₂,—O—P(═O)(—OH)(—O—(C₁-C₆) alkyl), —O—P(═O)(—O—(C₁-C₆) alkyl)₂,—O—P(═O)(—OH) (—O—(CH₂)-phenyl), —O—P(═O)(—O—(CH₂)-phenyl)₂, acarboxylic acid group, an amino carboxylic acid group and a peptide;

Q is selected from the group consisting of aryl, heteroaryl, —O-aryl,—S-aryl, —O-heteroaryl, and —S-heteroaryl;

X is selected from the group consisting of —(CH₂)—, —(NR8)-, S, and O;

R8 is independently selected from the group consisting of hydrogen,—(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉) cycloalkyl,—(C₃-C₉) substituted cycloalkyl, and —O—(C₁-C₆) alkyl, —C(═O)—O—(C₁-C₆)alkyl and —C(═O)—O—(C₁-C₆) substituted alkyl;

Y is selected from the group consisting of —(CH₂)— or a bond;

wherein said aryl, heteroaryl, —O-aryl, —S-aryl, —O-heteroaryl, and—S-heteroaryl groups may be substituted with one or more substituentsindependently selected from the group consisting of F, Cl, Br, I,—NR5R6, —(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉) cycloalkyl,—(C₃-C₉) substituted cycloalkyl, —O—(C₁-C₆) alkyl, —O—(C₁-C₆)substituted alkyl, —O—(C₃-C₉) cycloalkyl, —O—(C₃-C₉) substitutedcycloalkyl, -aryl, -aryl-(C₁-C₆) alkyl, -aryl-O —(C₁-C₆) alkyl, —O-aryl,—O—(C₁-C₄) alkyl-aryl, heteroaryl, heterocyclyl, —O—(C₁-C₄)alkyl-heterocycle, and —(S(═O)₂)—(C₁-C₆) alkyl; and

m is 1 or 2.

2.1. Definitions

The term “alkyl” refers to radicals containing carbon and hydrogen,without unsaturation. Alkyl radicals can be straight or branched.Exemplary alkyl radicals include, without limitation, methyl, ethyl,propyl, isopropyl, hexyl, t-butyl, sec-butyl and the like. Alkyl groupsmay be denoted by a range, thus, for example, a (C₁-C₆) alkyl group isan alkyl group having from one to six carbon atoms in the straight orbranched alkyl backbone. Substituted and unsubstituted alkyl groups mayindependently be (C₁-C₅) alkyl, (C₁-C₆) alkyl, (C₁-C₁₀) alkyl, (C₃-C₁₀)alkyl, or (C₅-C₁₀) alkyl. Unless expressly stated, the term “alkyl” doesnot include “cycloalkyl.”

A “cycloalkyl” group refers to a cyclic alkyl group having the indicatednumber of carbon atoms in the “ring portion,” where the “ring portion”may consist of one or more ring structures either as fused, spiro, orbridged ring structures. For example, a C₃ to C₆ cycloalkyl group (e.g.,(C₃-C₆) cycloalkyl) is a ring structure having between 3 and 6 carbonatoms in the ring. When no range is given, then cycloalkyl has betweenthree and nine carbon atoms ((C₃-C₉) cycloalkyl) in the ring portion.Exemplary cycloalkyl groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and adamantyl.Preferred cycloalkyl groups have three, four, five, six, seven, eight,nine, or from three to nine carbon atoms in the ring structure.

The term substituted alkyl and substituted cycloalkyl, refer to alkyland cycloalkyl groups, as defined above, substituted with one or moresubstituents independently selected from the group consisting offluorine, aryl, heteroaryl, —O—(C₁-C₆) alkyl, and —NR5R6, where R5 andR6 are independently selected from the group consisting of hydrogen and—(C₁-C₆) alkyl.

The term “aryl” refers to an aromatic carbocyclic group, having one,two, or three aromatic rings. Exemplary aryl groups include, withoutlimitation, phenyl, naphthyl, and the like. Aryl groups include one,two, or three aromatic rings structures fused with one or moreadditional nonaromatic carbocyclic or heterocyclic rings having from 4-9members. Examples of fused aryl groups include benzocyclobutanyl,indanyl, tetrahydronapthylenyl, 1,2,3,4-tetrahydrophenanthrenyl,tetrahydroanthracenyl, 1,4-dihydro-1,4-methanonaphthalenyl,benzodioxolyl.

The term “heteroaryl” refers to a heteroaromatic (heteroaryl) grouphaving one, two, or three aromatic rings containing from 1-4 heteroatoms(such as nitrogen, sulfur, or oxygen) in the aromatic ring. Heteroarylgroups include one, two, or three aromatic rings structures containingfrom 1-4 heteroatoms fused with one or more additional nonaromatic ringshaving from 4-9 members. Heteroaryl groups containing a single type ofheteroatom in the aromatic ring are denoted by the type of hetero atomthey contain, thus, nitrogen-containing heteroaryl, oxygen-containingheteroaryl and sulfur-containing heteroaryl denote heteroaromatic groupscontaining one or more nitrogen, oxygen or sulfur atoms respectively.Exemplary heteroaryl groups include, without limitation, pyridyl,pyrimidinyl, triazolyl, quinolyl, quinazolinyl, thiazolyl,benzo[b]thiophenyl, furanyl, imidazolyl, indolyl, and the like.

The terms “heterocyclyl” or “heterocycle” refers to either saturated orunsaturated, stable non-aromatic ring structures that may be fused,spiro or bridged to form additional rings. Each heterocycle consists ofone or more carbon atoms and from one to four heteroatoms selected fromthe group consisting of nitrogen, oxygen and sulfur. “Heterocyclyl” or“heterocycle” include stable non-aromatic 3-7 membered monocyclicheterocyclic ring structures and 8-11 membered bicyclic heterocyclicring structures. A heterocyclyl radical may be attached at anyendocyclic carbon or nitrogen atom that results in the creation of astable structure. Preferred heterocycles include 3-7 membered monocyclicheterocycles (more preferably 5-7-membered monocyclic heterocycles) and8-10 membered bicyclic heterocycles. Examples of such groups includepiperidinyl, piperazinyl, pyranyl, pyrrolidinyl, morpholinyl,thiomorpholinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl,isoxozolyl, tetrahydropyranyl, tetrahydrofuranyl, dioxolyl, dioxinyl,oxathiolyl, dithiolyl, sulfolanyl, dioxanyl, dioxolanyl,tetahydrofurodihydrofuranyl, tetrahydropyranodihydro-furanyl,dihydropyranyl, tetrahydrofurofuranyl, tetrahydropyranofuran,quinuclidinyl (1-azabicyclo[2.2.2]octanyl) and tropanyl(8-methyl-8-azabicyclo[3.2.1]octanyl).

For the purpose of the Q subsituent, the term “substituted 3-indolyl”refers to a 3-indolyl group substituted with one or more substituentsselected from the group consisting of: F, Cl, Br, I, —NR5R6, —(C₁-C₆)alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉) cycloalkyl, —(C₃-C₉)substituted cycloalkyl, —O—(C₁-C₆) alkyl, —O—(C₁-C₆) substituted alkyl,—O—(C₃-C₉) cycloalkyl, —O—(C₃-C₉) substituted cycloalkyl, -aryl,-aryl-(C₁-C₆) alkyl, -aryl-O—(C₁-C₆) alkyl, —O-aryl, —O—(C₁-C₄)alkyl-aryl, heteroaryl, heterocyclyl, —O—(C₁-C₄) alkyl-heterocycle, and—(S(═O)₂)—(C₁-C₆) alkyl; where R5, R6 are independently selected fromthe group consisting of hydrogen, and —(C₁-C₆) alkyl.

For the purposes of the R7 substituent, the term “carboxylic acid group”refers to a group of the form —O—C(═O)—(C₁-C₆) alkyl, —O—C(═O)—(C₃-C₉)cycloalkyl, —O—C(═O)-aryl, —O—C(═O)-heteroaryl, —O—C(═O)-heterocycle,—O—C(═O)—(C₁-C₆) alkyl-aryl, —O—C(═O)—(C₁-C₆) alkyl-heteroaryl, or—O—C(═O)—(C₁-C₆) alkyl-heterocycle. Included in “carboxylic acid group”are groups of the form —O—C(═O)—(C₁-C₆) alkyl, —O—C(═O)—(C₃-C₉)cycloalkyl, —O—C(═O)-aryl, —O—C(═O)-heteroaryl, —O—C(═O)-heterocycle,—O—C(═O)—(C₁-C₆) alkyl-aryl, —O—C(═O)—(C₁-C₆) alkyl-heteroaryl, or—O—C(═O)—(C₁-C₆) alkyl-heterocycle substituted with one or moresubstituent independently selected from the group consisting of: F, Cl,Br, I, —OH, —SH, —NR5R6, —(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl,—(C₃-C₉) cycloalkyl, —(C₃-C₉) substituted cycloalkyl, —O—(C₁-C₆) alkyl,—O—(C₁-C₆) substituted alkyl, —S—(C₁-C₆) alkyl, —O—(C₃-C₉) cycloalkyl,—O—(C₃-C₉) substituted cycloalkyl, -aryl, —O-aryl, —O—(C₁-C₄)alkyl-aryl, heteroaryl, heterocyclyl, —O—(C₁-C₄) alkyl-heterocycle,—(S(═O)₂)—(C₁-C₆) alkyl, —NH—C(═NH)—NH₂ (i.e., guanido), —COOH, and—C(═O)—NR5R6, where R5 and R6 are independently selected from the groupconsisting of hydrogen, and —(C₁-C₆) alkyl. In addition, for thepurposes of the R7 substituent the term “amino carboxylic acid group”refers to a carboxylic acid group, including carboxylic acid groupssubstituted with one or more of the above-stated substituents, whichbears one or more independently selected amino groups of the form —NR5R6where R5 and R6 are independently selected from the group consisting ofhydrogen and (C₁-C₆) alkyl.

In one embodiment of this invention, R7 is an alpha amino or imino acid,including but not limited to alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine or stereoisomers or racemicmixtures thereof. In another embodiment the of the invention, R7 isalpha amino or imino acid selected from the group consisting ofL-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine,L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine,L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, and L-valine.

For the purposes of the R7 substituent, the term “peptide” refers to adipeptide, tripeptide, tetrapeptide or pentapeptide, which release two,three, four, or five amino or imino acids (e.g., proline) respectivelyupon hydrolysis. For the purpose of R7, peptides are linked to theremainder of the molecule through an ester linkage. In one embodiment,peptides of R7 are comprised of alpha amino or imino acid, including butnot limited to alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, valine or stereoisomers or racemic mixturesthereof; and in a more preferred version of this embodiment, thecarboxyl group involved in the ester linkage is the carboxyl terminalCOOH group of the peptide, as opposed to a side chain carboxyl. Inanother embodiment the of the invention, R7 is alpha amino or imino acidselected from the group consisting of L-alanine, L-arginine,L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid,L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and L-valine; and in a more preferred version of thispreferred embodiment, the carboxyl group involved in the ester linkageis the carboxyl terminal COOH group of the peptide, as opposed to a sidechain carboxyl.

2.2. Preferred Compounds

Included in the preferred embodiments are compounds of formula III,IIIa, IVa, IVb, Va, or Vb, wherein Q is selected from the groupconsisting of aryl, heteroaryl, —O-aryl, —S-aryl, —O-heteroaryl, and—S-heteroaryl, provided that Q is not 3-indolyl or a substituted3-indolyl. In other preferred embodiments Q is selected from the groupconsisting of aryl, heteroaryl, —O-aryl, —S-aryl, —O-heteroaryl, and—S-heteroaryl, provided that when R4 is hydrogen, cycloalkyl, or alkyl,Q is not 3-indolyl or a substituted 3-indolyl. In still other preferredembodiments Q is selected from the group consisting of aryl, heteroaryl,—O-aryl, —S-aryl, —O-heteroaryl, and —S-heteroaryl, provided that whenR4 is hydrogen, (C₃-C₄) cycloalkyl, or (C₁-C₄) alkyl, Q is not 3-indolylor substituted 3-indolyl. In another preferred embodiment Q is 3-indolylor a substituted 3-indolyl provided that R4 is not hydrogen, cycloalkyl,or alkyl. In still another preferred embodiment Q is 3-indolyl or asubstituted 3-indolyl provided that R4 is not hydrogen, (C₃-C₄)cycloalkyl, or (C₁-C₄) alkyl.

Other preferred embodiments include compounds of formula III, IIIa, IVa,IVb, Va, or Vb where R4 is —CH₂R7. These compounds may serve as prodrugforms of the corresponding compounds of formula III, IIIa, IVa, IVb, Va,or Vb where R4 is H. The prodrug form is cleaved by hydrolysis torelease the corresponding compound where R4 is H. The hydrolysis mayoccur by enzymatic or nonenzymatic routes that produce the correspondinghydroxymethylene derivative, which upon subsequent hydrolysis, result inthe release of compounds where R4 is H. In one such preferred embodimentR4 is —CH₂R7, where R7 is —O—P(═O)(OH)₂, —O—P(═O)(—OH)(—O—(C₁-C₆)alkyl),or —O—P(═O)(—O—(C₁-C₆)alkyl)₂. In one embodiment where R7 is—O—P(═O)(—O—(C₁-C₆)alkyl)₂, the alkyl groups are independently selected.In another preferred embodiment, R4 is —CH₂R7, where R7 is a carboxylicacid group or an amino carboxylic acid group. In still another preferredembodiment R7 is a peptide; where in a more preferred embodiment thepeptide is linked through an ester bond formed with the carboxylterminal COOH group of the peptide chain to the remainder of thecompound. In other preferred separate and independent embodiments ofcompounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is —CH₂R7and R7 is a peptide, the peptide may be a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Preferred amino acid compositions forpeptides of the R7 functionality are described above.

Embodiments of compounds of formula III, IIIa, IVa, IVb, Va, or Vbinclude those where X is selected from the group consisting of —(NR8)-,S, and O, where R8 is independently selected from the group consistingof hydrogen, —(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉)cycloalkyl, —(C₃-C₉) substituted cycloalkyl, and —O—(C₁-C₆) alkyl. Otherembodiments of compounds of formula III, IIIa, IVa, IVb, Va, or Vbinclude those where X is —CH₂—. In other embodiments of compounds offormula III, IIIa, IVa, IVb, Va, or Vb, X is oxygen (O). In otherembodiments of compounds of formula III, IIIa, IVa, IVb, Va, or Vb, X issulfur (S). In still other embodiments of compounds of formula III,IIIa, IVa, IVb, Va, or Vb, X is —(NR8)-, where R8 is independentlyselected from the group consisting of hydrogen, —(C₁-C₆) alkyl, —(C₁-C₆)substituted alkyl, —(C₃-C₉) cycloalkyl, —(C₃-C₉) substituted cycloalkyl,and —O—(C₁-C₆) alkyl.

Other preferred embodiments of the invention include compounds offormula III or Ma, where Q is a heteroaryl or an optionally substitutedheteroaryl group. In four separate alternative preferred embodiments ofcompounds of formula III or IIIa, Q is an optionally substitutedmonocyclic heteroaryl group, an optionally substituted bicyclicheteroaryl group, an optionally substituted bicyclic heteroaryl groupwith the proviso that the bicyclic heteroaryl group is not an indolylgroup or a substituted indolyl, or an optionally substituted tricyclicheteroaryl group. Optional substituents, when present, are independentlyselected from those recited for aryl, heteroaryl, —O-aryl, —S-aryl,—O-heteroaryl, and —S-heteroaryl.

Included in the preferred embodiments of the invention are compounds offormula IVa, IVb, Va, or Vb, where Q is a heteroaryl or an optionallysubstituted heteroaryl group. In four separate alternative preferredembodiments of compounds of formula IVa, IVb, Va, or Vb, Q is anoptionally substituted monocyclic heteroaryl group, an optionallysubstituted bicyclic heteroaryl group, an optionally substitutedbicyclic heteroaryl group with the proviso that the bicyclic heteroarylgroup is not indolyl, or an optionally substituted tricyclic heteroarylgroup. In a more preferred embodiment, Q is an optionally substitutednitrogen-containing heteroaryl group. In a related embodiment, Q is anoptionally substituted indolyl. Optional substituents, when present areindependently selected from those recited for aryl, heteroaryl, —O-aryl,—S-aryl, —O-heteroaryl, and —S-heteroaryl.

Preferred embodiments of the invention include mixtures of compounds offormula IVa and IVb, including racemic mixtures. In another preferredembodiment, the compounds of formula IVa and IVb are the separateenantiomers of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.In this embodiment the preparation of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis prepared as a mixture beginning with the starting materials1,2,3,4-tetrahydroquinoline and indole-3-acetamide. The1,2,3,4-tetrahydroquinoline is converted into5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acid methylester as described in Example 1, steps 1-5. The5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acid methylester is reacted with indole-3-acetamide as described in Example 1, step6, to yield3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione.The mixture of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis then prepared by catalytic hydrogenation as described in Example 2using Procedure B.

Preferred embodiments of the invention also include mixtures ofcompounds of formula Va and Vb, including racemic mixtures. In anotherpreferred embodiment, the compounds of Va and Vb are the separateenantiomers of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.In this embodiment, the compounds are prepared as a mixture by firstpreparing(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,as described above. The mixture of cis compounds is then treated with amixture of potassium tert-butoxide in tert-butanol to obtain a mixtureof(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneas described in Example 3.

All stereoisomers of the compounds of the instant invention arecontemplated, either in admixture or in pure or substantially pure form,including crystalline forms of racemic mixtures and crystalline forms ofindividual isomers. The definition of the compounds according to theinvention embraces all possible stereoisomers (e.g., the R and Sconfigurations for each asymmetric center) and their mixtures. It veryparticularly embraces the racemic forms and the isolated optical isomershaving a specified activity. The racemic forms can be resolved byphysical methods, such as, for example, fractional crystallization,separation or crystallization of diastereomeric derivatives, separationby chiral column chromatography or supercritical fluid chromatography.The individual optical isomers can be obtained from the racemates byconventional methods, such as, for example, salt formation with anoptically active acid followed by crystallization. Furthermore, allgeometric isomers, such as E- and Z-configurations at a double bond, arewithin the scope of the invention unless otherwise stated. Certaincompounds of this invention may exist in tautomeric forms. All suchtautomeric forms of the compounds are considered to be within the scopeof this invention unless otherwise stated. The present invention alsoincludes one or more regioisomeric mixtures of an analog or derivative.

As used herein, the term “salt” is a pharmaceutically acceptable saltand can include acid addition salts including hydrochlorides,hydrobromides, phosphates, sulphates, hydrogen sulphates,alkylsulphonates, arylsulphonates, acetates, benzoates, citrates,maleates, fumarates, succinates, lactates, and tartrates; alkali metalcations such as Na⁺, K⁺, Li⁺, alkali earth metal salts such as Mg or Ca,or organic amine salts.

As used herein, the term “metabolite” means a product of metabolism of acompound of formula III, IIIa, IVa, IVb, Va, or Vb, or apharmaceutically acceptable salt, analog or derivative thereof, thatexhibits a similar activity in vivo to said a compound of formula III,IIIa, IVa, IVb, Va, or Vb.

As used herein, the term “prodrug” means a compound of formula III,IIIa, IVa, IVb, Va, or Vb covalently linked to one or more pro-moieties,such as an amino acid moiety or other water solubilizing moiety. Acompound of formula III, IIIa, IVa, IVb, Va, or Vb may be released fromthe pro-moiety via hydrolytic, oxidative, and/or enzymatic releasemechanisms. In an embodiment, a prodrug composition of the presentinvention exhibits the added benefit of increased aqueous solubility,improved stability, and improved pharmacokinetic profiles. Thepro-moiety may be selected to obtain desired prodrug characteristics.For example, the pro-moiety, e.g., an amino acid moiety or other watersolubilizing moiety such as phosphate within R4, may be selected basedon solubility, stability, bioavailability, and/or in vivo delivery oruptake.

3. The Synthesis of Pyrroloquinolinyl-pyrrole-2,5-diones andpyrroloquinolinyl-pyrrolidine-2,5-diones

Standard synthetic methods and procedures for the preparation of organicmolecules and functional group transformations and manipulationsincluding the use of protective groups can be obtained from the relevantscientific literature or from standard reference textbooks in the field.Although not limited to any one or several sources, recognized referencetextbooks of organic synthesis include: Smith, M. B.; March, J. March'sAdvanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5^(th)ed.; John Wiley & Sons: New York, 2001; and Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 3^(rd); John Wiley & Sons:New York, 1999. The following descriptions of synthetic methods aredesigned to illustrate, but not limit, general procedures for thepreparation of the compounds of formula III, IIIa, IVa, IVb, Va, or Vb.

3.1 General Procedures for the Synthesis ofpyrroloquinolinyl-pyrrole-2,5-dione andpyrroloquinolinyl-pyrrolidine-2,5-diones where R4 is hydrogen

The present invention provides for pyrroloquinolinyl-pyrrole-2,5-dionecompounds of formula III, IIIa, IVa, IVb, Va, or Vb. The preparation ofcompounds of formula III, IIIa, IVa, IVb, Va, and Vb may be achieved bya series of reactions commencing with the reaction of a5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acid ester offormula I with an amide of formula II, to form a3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dioneof formula III, including compounds of formula Ma, where R4 is hydrogen,as shown in Scheme 1.

3.1.1. Synthesis of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dionesof formula III where R4 is hydrogen

The condensation of an ester of formula I and a compound of formula IIto produce compounds of formula III, including compounds of formula Ma,where R4 is hydrogen is conducted in any suitable anhydrous polaraprotic solvent including, but not limited to, tetrahydrofuran (THF),tetrahydropyran, diethyl ether and the like in the presence of base. Forthe purposes of the reaction, suitable esters of formula I include, butare not limited to, alkyl esters where R9 is a (C1-C4) alkyl group, andpreferred esters include the methyl and ethyl esters. Suitable bases forthe reaction include alkaline metal salts of low molecular weight alkylalcohols, including, but not limited to, alkaline metal salts ofmethanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, andtert-butanol. Preferred alkaline metal salts of low molecular weightalkyl alcohols include sodium and potassium salts, with potassiumtert-butoxide (tBuOK) being the preferred base. Typically the reactionsare conducted at 0° C. for 2 hours, however, both the time andtemperature may be altered depending upon the specific substituentspresent on compounds of formula I and II, and the solvent employed. Thereaction temperature may be varied from −78° C. to 37° C., and ispreferably from −35° C. to 25° C., or more preferably from −15° C. to10° C. Reaction times will generally vary inversely with the temperatureemployed, suitable times from about 15 minutes to 24 hours may beemployed, more preferably, 30 minutes to 12 hours, and more preferably 1to 6 hours.

3.1.2. Preparation of Compounds of Formula IVa, IVb, Va and Vb where R4is hydrogen

Reduction of compounds of formula III and Ma, where R4 is hydrogen toyield the corresponding3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneshaving formula IVa, IVb, Va, or Vb may be conducted employing a varietyof procedures including, but not limited to, reduction with zinc-mercury(Procedure A), catalytic hydrogenation (Procedure B), and reduction withmagnesium in methanol (Procedure C). As indicated in Scheme 1, dependingon the reduction reaction and conditions chosen, the reaction will yieldprincipally compounds of formula IVa and IVb, or principally compoundsof formula Va and Vb, or alternatively a mixture of compounds of formulaIVa, IVb, Va, and Vb.

Mixtures of compounds of formula IVa, IVb, Va, and Vb may be prepared bythe direct reduction of compounds of formula III or IIIa with azinc-mercury reducing agent. The reaction is generally carried out withfresh reducing agent prepared by mixing Zn powder with HgCl₂ deionizedwater followed by acidification with HCl. After drying, the solidreducing agent (zinc-mercury) is suitable for reduction of compounds offormula III or Ma in refluxing dry ethanol under a dry HCl gasatmosphere as described in Example 2, Procedure A, for the reduction of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione.

An alternative method of preparing pyrrolidine-2,5-diones is catalytichydrogenation, which yields a mixture consisting principally of the(±)-cis pyrrolidine-2,5-diones of formula IVa and IVb. Catalytichydrogenation of compounds of formula III or IIIa may be conducted in ananhydrous alcohol over a noble metal catalyst under 1 atmosphere ofhydrogen for 48 hours. A variety of low molecular weight alkyl alcoholsmay be employed to conduct the reduction, including n-propyl alcohol,isopropyl alcohol, ethanol or methanol. Preferably the alcohol isethanol or methanol, and most preferably methanol. A noble metalcatalyst (e.g., platinum, palladium, rhodium, ruthenium etc.) oncharcoal is preferred for the reduction of compounds of formula III orIIIa. In more preferred embodiments, the noble metal catalyst ispalladium on activated charcoal. While reduction compounds of formulaIIIa or III under 1 atmosphere of hydrogen at room temperature (25° C.)for 12-48 hours is generally suitable for preparation ofpyrrolidine-2,5-diones, the pressure of hydrogen, reaction time, and thereaction temperature may be varied. Catalytic hydrogenation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dioneis described in Example 2, Procedure B.

Pyrrole-2,5-diones of formula Ma or III may be reduced to yield amixture of compounds of formula Va and Vb by the reduction in anhydrousalcohol with a metal reducing agent. Preferred metals include sodium,calcium and magnesium, with magnesium as a more preferred metal reducingagent. The reaction is typically carried out under an inert atmosphereof nitrogen for 30 minutes to 2 hours by refluxing a compound of formulaIII or formula IIIa in an alcohol selected from the group consisting ofmethanol, ethanol, n-propanol, and isopropanol with magnesium turnings.In preferred embodiments the reaction is conducted for about 40 minutesin methanol as described in Example 2, Procedure C, for the preparationof(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Compounds of IVa and/or IVb, which have the pyrrolidine ringsubstituents in the cis configuration, may be converted into a mixtureof compounds of Va and Vb, where the substituents are in the transconfiguration, or into a mixture of all four isomers of formula IVa,IVb, Va, and Vb by treatment with base in a polar protic solvent.Typically the reaction employs an alkaline metal salt of a (C1-C4) alkylalcohol in an alcohol solvent (e.g., sodium or potassium methoxide inmethanol, sodium or potassium ethoxide in ethanol, sodium or potassiumtert-butoxide in tert-butanol), with potassium tert-butoxide intert-butanol as the preferred alkaline metal salt and solvent mixture.Reactions are generally conducted from 0° C. to the reflux temperatureof the reaction mixture for 4 to 48 hours. In more preferredembodiments, the reaction are conducted from room temperature (25° C.)to the reflux temperature of the mixture for 8 to 24 hours, and in aneven more preferred embodiment, the reaction is conducted at about 50°C. in a mixture of potassium tert-butoxide in tert-butanol for about 16hours. Short reaction times and low temperatures favor formation ofmixtures still containing compounds IVa and/or IVb.

3.1.3. Introduction of Aryl or Heteroaryl Substituents into Compounds ofIII, IIIa, IVa, IVb, Va, and Vb

The introduction of additional substituted and unsubstituted aryl orheteroaryl substituents on to aromatic rings of compounds of formulaIII, IIIa, IVa, IVb, Va, or Vb may be accomplished by the reaction of asubstituted or unsubstituted aryl or heteroaryl boronic acid with anaromatic halogen substituent on a compound of formula III, IIIa, IVa,IVb, Va, or Vb. The reaction is typically carried out by heating amixture of a compound of formula III, IIIa, IVa, IVb, Va, or Vb bearingan aryl or heteroaryl bromide or iodide, more preferably an arylbromideor hetroarylbromide, with an aryl or heteroaryl boronic acid in thepresence of tetrakistriphenylphosphine palladium in a solvent mixtureconsisting of 5 parts toluene, 5 parts ethanol, 1 part saturated NaHCO₃,and 2 parts water to 100° C. under nitrogen for 5 hours. After coolingto room temperature, the mixture is extracted with ethyl acetate andconcentrated. The residue is purified by silica gel chromatography. In apreferred embodiment, the halogenated compound of formula III, IIIa,IVa, IVb, Va, or Vb bears the halogen on an aryl or heteroaryl group Qfunctionality resulting in the introduction of substituted aryl orheteroaryl group donated by the boronic acid on to the Q substituent. Ina more preferred embodiment, the Q functionality is a brominatedaromatic or heteroaromatic Q functionality. In another more preferredembodiment the halogenated Q functionality reacted with the boronic acidis a halogenated 3-indolyl. Examples 31-34 describe the introduction ofsubstituted and unsubstituted aromatic groups into compounds of formulaVa and Vb employing a brominated Q functionality where Q is a brominated3-indolyl.

Aromatic and heteroaromatic boronic acids including 2-thienylboronicacid, 3-thienylboronic acid, and 2-naphthylboronic acid are availablefrom a variety of commercial sources including Sigma-Aldrich (St. Louis,Mo.). Alternatively aromatic and heteroaromatic boronic acids may beprepared from the corresponding aryl or heteroaryl bromides by reactionwith triisopropyl borate in the presence of n-butyllithium followed byquenching with aqueous HCl. (See, e.g., W. Li, et. al., J. Organic Chem.67: 5394-97 (2002) and C. M. Marson, et. al., Tetrahedron 59: 4377-81(2003).

3.1.4. Preparation of Compounds of Formula III, IIIa, IVa, IVb, Va, andVb where R4 is —CH₂R7

Compounds of formula III, IIIa, IVa, IVb, Va, or Vb, where R4 ishydrogen, can be converted into compounds of formula III, IIIa, IVa,IVb, Va, or Vb where R4 is —CH₂R7. The conversion begins with thepreparation of the hydroxymethylene derivative of the compounds asindicated in the partial structures shown in Scheme 2.

Preparation of the hydroxymethylene derivatives is accomplished byreaction of a compound of formula III, IIIa, IVa, IVb, Va, or Vb whereR4 is H with aqueous formaldehyde in tetrahydrofuran (THF). Typicalreaction conditions employ equal volumes of THF and 37% formaldehyde inwater with the reaction stirred for 14-16 hours at room temperature.Reaction times and temperatures may vary from 1 hour to 48 hours and thetemperature may be from 0° C. to 50° C. or more preferably from 10° C.to 37° C. Upon completion the reaction is partitioned between water andan organic solvent, typically ethyl acetate. The organic layer is driedover sodium sulfate, concentrated, and subject to chromatography onsilica gel as necessary to yield the hydroxymethylene product. Thepreparation of the hydroxymethylene derivative of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione,is described in Example 56, step 1.

Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is —CH₂R7and R7 is phosphate (—O—P(═O)(OH)₂), monoalkyl phosphate (e.g.,—O—P(═O)(—OH)(—O—(C₁-C₆) alkyl)), dialkyl phosphate (e.g.,—O—P(═O)(—O—(C₁-C₆) alkyl)₂) a monobenzylphosphate ester (—O—P(═O)(—OH)(—O—(CH₂)-phenyl)), or a dibenzylphosphate ester(—O—P(═O)(—O—(CH₂)-phenyl)₂) may be prepared from the desiredhydroxymethylene derivative and a suitably substituted phosphoric acidby any reaction suitable for the formation of a phosphate ester bondbetween the phosphoric acid compound and the hydroxymethylenederivative. In a preferred method, the formation of phosphate esters isconducted by reaction of a hydroxymethylene derivative of a compound offormula III, IIIa, IVa, IVb, Va, or Vb with a suitably protectedphosphoramidate followed by deprotection. Reactions with the desiredphosphoramidate are typically conducted at room temperature in anhydrousTHF. Following the addition of the phosphoramidate, the reaction istreated with tetrazole (3% in acetonitrile) and stirred 5 minutes to 1hour, after which the reaction is cooled to −78° C. The cooled reactionis treated with m-chloroperbenzoic acid, and after stirring at −78° C.for 5 minutes, the reaction is warmed to room temperature and stirredfor 5 minutes further. Following the removal of solvent, the product ispurified by flash chromatography on silica gel using ethyl acetatehexane. The protecting groups are removed by suitable deprotectionreactions. Where the phosphoramidate employed isdibenzylphosphoramidate, the benzyl protecting groups may be removed byhydrogenation of the compound over Pd/C under 1 atmosphere of hydrogenat room temperature. The preparation of phosphoric acidmono-[3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethyl]esterfrom3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dioneis described in Example 56, steps 2-3.

Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R7 is acarboxylic acid group, or an amino carboxylic acid group, may beprepared by coupling the desired hydroxymethylene derivative with acarboxylic acid or amino carboxylic acid (amino acid) under conditionssuitable for the formation of an ester linkage. A variety of dehydratingagents, including DCC (dicyclohexylcarbodiimide), HBTU(0-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), or BOP ((benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate) may be employed to drive the formationof the ester bond. In a preferred embodiment, the reactions areconducted in anhydrous THF in the presence of HBTU and DIEPA(N,N-diisopropylethylamine) at room temperature for 10 hours to 24hours. Following completion of the dehydration reaction, solvent isremoved under reduced pressure and the compounds are taken up in anorganic solvent (e.g., ethyl acetate) and washed with water. The organiclayer is dried and the residue purified by silica gel chromatography asnecessary.

Where R7 is an amino carboxylic acid group, the starting materials forintroducing the amino carboxylic acid group must contain a suitablyprotected amine A variety of suitable amine-protecting groups may beadvantageously employed including carbobenzyloxy-protected amines (e.g.,the reactions may employ N-carbobenzyloxy glycine or N-carbobenzyloxyalanine etc.). Subsequent deprotection will yield the free product.Where the protecting group employed is carbobenzyloxy, deprotection maybe accomplished by treating the amine protected product suspended inmethanol with HCl (4M) in ethyl acetate in the presence of palladium oncharcoal (Pd/C) under 1 atmosphere of hydrogen for 1-3 hours at roomtemperature. Examples 58-60 describe the preparation of compounds whereR7 is a carboxylic acid group, or an amino carboxylic acid group.

Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R7 is apeptide, may be prepared by coupling the desired hydroxymethylenederivative with a peptide bearing a free carboxylic acid group to forman ester linkage. Linking of a carboxyl functionality of a peptide andthe hydroxymethylene group in an ester linkage may be conductedemploying a suitably protected peptide, bearing for example, protectedfree amine groups protected with conventional N-protecting groups.Conditions suitable for the formation of an ester linkage, include thoseemploying dehydrating agents, such as those described for thepreparation of compounds of formula III, IIIa, IVa, IVb, Va, or Vb whereR4 is —CH₂R7 and R7 is a carboxylic acid group, or an amino carboxylicacid group.

3.1.5. Preparation of Compounds of Formula III, IIIa, IVa, IVb, Va, andVb where R4 is —(C₁-C₆) alkyl

Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is a—(C₁-C₆) alkyl may be prepared by reacting by reacting the desiredcompound of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is H with a(C₁-C₆) alkyl halide, where the halide is preferably Cl, Br or I, in thepresence of a suitable base at room temperature. Suitable bases includeorganic bases such as potassium tert-butoxide, sodium methoxide, andinorganic bases such as KOH, NaOH and K₂CO₃. Suitable solvents includepolar aprotic solvents such as DMSO, THF, dioxane or other ethers, orDMF. In an alternative embodiment, the compound of formula III, IIIa,IVa, IVb, Va, or Vb where R4 is H is reacted with an organic orinorganic base to yield the conjugate base of the compound of formulaIII, IIIa, IVa, IVb, Va, or Vb, and the conjugate base is then reactedwith the alkylhalide. Where the alkyl group is introduced into acompound of formula III or IIIa, the resulting alkylated compounds canbe reduced to yield compounds of formula IVa and IVb, Va and Vb, or amixture of compounds of formula IVa, IVb, Va, and Vb employing thereduction procedures described in Section I(b)(1). Example 61 describesthe preparation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrole-2,5-dioneusing iodomethane as an alkylating agent, and its reduction by catalytichydrogenation to yield(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrolidine-2,5-dione.

Compounds of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is a—(C₁-C₆) alkyl group may also be prepared by reacting the desiredcompound of formula III, IIIa, IVa, IVb, Va, or Vb where R4 is H with a(C₁-C₆) alkyl alcohol in the presence of diethylzodicarboxylate (DEAD)and triphenylphosphine. (See, e.g., Mitsunobu, O.; Wade, M.; Sano, T. J.Am Chem. Soc. 94: 694 (1972); Hughes, D. L., Organic Reactions, 42;335-656 (1992)). The reactions may be conducted in a variety of solventsincluding tetrahydrofuran (THF), dichloromethane, chloroform,acetonitrile, and benzene, preferably the solvent is THF.

3.1.6. Separation of Compounds of Formula III, IIIa, IVa, IVb, Va, andVb

Where the isolation of an individual product having the structure offormula III, IIIa, IVa, IVb, Va, or Vb is desired, the products may beseparated by chromatography on one or more chromatography media.Chromatography may be carried out on a preparative scale or on ananalytical scale to determine the identity and purity of the productspresent in a sample. Although any suitable chromatography mediaincluding, but not limited to, silica, reverse phase, ion exchange,chiral chromatographic media, or any combination thereof, may beadvantageously employed for separations, the suitability of specificchromatographic media and conditions for the separation of productshaving formula III, IIIa, IVa, IVb, Va, and Vb will depend upon thesubstituents present on the compounds. In preferred embodiments,chromatographic separations are conducted employing HPLC. In otherpreferred embodiments the separation is carried out using supercriticalfluid chromatography. Where supercritical fluid chromatography isemployed, CO₂, or mixtures of CO₂ with other solvents includingacetonitrile (ACN), methanol, ethanol, isopropanol, or hexane, are thepreferred mobile phase, with mixtures of CO₂ and methanol mostpreferred. A variety of chromatographic media (stationary phases) may beemployed in supercritical fluid chromatography including, but notlimited to: ChiralCel OA, OB, OD, or OJ; ChiralPak AD or AS; CyclobondI, II, or III; and Chirobiotic T, V, and R media.

In more preferred embodiments, where the products are individual isomersof formula IVa, IVb, Va, or Vb, mixtures containing two or more of theisomeric forms may be separated by using supercritical fluidchromatography on chiral media. In one more preferred embodiment,separations are conducted on CHIRALPAK® AD columns (Daicel (U.S.A.) Inc.Fort Lee, N.J.). In that embodiment, products are applied to the ADcolumn in a mixture of methanol and acetonitrile, or in acetonitrile,and the column is subsequently eluted with 35% methanol in CO2 (65%).The separation of3(R),4(S)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand3(S),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneon a CHIRALPAK® AD column is set forth in Example 4. The separation of(+)trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand (−)trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis set forth in Example 5.

The individual racemic forms of compounds of formula III, IIIa, IVa,IVb, Va, or Va may also be resolved by physical methods, such as, forexample, fractional crystallization or crystallization of diastereomericderivatives. In addition, individual optical isomers can be obtainedfrom racemic mixtures by conventional methods, such as, for example,salt formation with an optically active acid, where applicable, followedby crystallization.

3.2. Preparation of Compounds of Formula I and II where Y is a Bond

Compounds of formula I and II, which are employed in the synthesis ofpyrroloquinolinyl-pyrrole-2,5-dione of formula III and Ma, may bepurchased or obtained via a variety of synthetic routes such as thoseset forth below.

3.2.1. Preparation of Compounds of Formula I where Y is a Bond

Compounds of formula I may be prepared from the corresponding compoundof formula A, where X is selected from the group consisting of —(CH₂)—,—(NR8)-, S and O, R8 is selected from the group consisting of hydrogen,—(C₁-C₆) alkyl, —(C₁-C₆) substituted alkyl, —(C₃-C₉) cycloalkyl,—(C₃-C₉) substituted cycloalkyl, and —O—(C₁-C₆) alkyl, and m is 1 or 2.Exemplary compounds of formula A include 1,2,3,4-tetrahydroquinoline,1,2,3,4-tetrahydro-quinoxaline, 3,4-dihydro-2H-benzo[1,4]oxazine,3,4-dihydro-2H-benzo[1,4]thiazine,2,3,4,5-tetrahydro-1H-benzo[b]azepine,2,3,4,5-tetrahydro-1H-benzo[b][1,4]diazepine,6,7,8,9-tetrahydro-5-oxa-9-aza-benzocycloheptene, or2,3,4,5-tetrahydro-benzo[b][1,4]thiazepine. The preparation begins withthe conversion of a compound of formula A to the corresponding3-substituted-2-oxopropionic acid ethyl ester of formula B. The ethylester of formula B is cyclized to form a compound of formula C, which isconverted to the free acid D, which is decarboxylated to yield thedesired tricyclic product E. Subsequent reaction of the tricyclicproduct E with oxalyl chloride and work-up in alcoholic base yields thecorresponding compound of formula I. Scheme 3 illustrates the reactionsequence beginning with compounds of formula A, which is furtherillustrated in Example 1, steps 1-5 for the preparation of5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic methyl ester offormula I from 1,2,3,4-tetrahydroquinoline and bromoethylpyrruvate(3-bromo-pyruvic acid ethyl ester).

Some suitable conditions for the conversion of compounds of formula Ainto compounds of formula I through the reaction sequence of Scheme 3are described herein. Compounds of formula A may be converted to thecorresponding 3-substituted-2-oxopropionic acid ethyl ester of formula Bby treatment with bromoethyl pyruvate in an anhydrous ether, such asTHF, at room temperature for about 24 hours. Treatment of the3-substituted-2-oxopropionic acid ethyl ester of formula B withanhydrous MgCl₂ in 2-methoxyethanol at about 125° C. for 30 minutes to 2hours, preferably for 1 hour, results in the formation of thecorresponding tricyclic carboxylic acid ester of formula C. Subsequentconversion of this compound to the free acid of formula D may beaccomplished by hydrolysis in aqueous base. In preferred embodiments thereaction is carried out in an aqueous base, including but not limited toNaOH or KOH, in the presence of alcohol as a co-solvent. Preferredalcohol co-solvents include methanol, ethanol, n-propanol, andisopropanol, with ethanol as a more preferred co-solvent. Reactions aretypically conducted by heating the mixture to reflux for 2 hours,although the time and temperature of the reaction may be varied asneeded. Oxidative decarboxylation of compounds of formula D may beconducted by a variety of procedures suitable for the decarboxylation ofaromatic acids. In preferred embodiments the decarboxylation ofcompounds of formula D is conducted by heating the free acid withcopper-chromite (CuO—Cr₂O₃) in quinolone for about 2 hours to yield thedecarboxylated product of formula E. Conversion of compounds of formulaE to compounds of formula I may be accomplished by reaction with oxalylchloride, followed by treatment with a mixture of an anhydrous alcoholand the alkaline metal salt of the alcohol, preferably sodium methoxide,or sodium ethoxide. The reaction of oxalyl chloride with compounds offormula E is typically conducted in anhydrous polar aprotic solventsincluding ethers at a temperature from about −78° C. to about 10° C. Inpreferred embodiments, the reaction is conducted at a temperature fromabout −25° C. to about 5° C. employing an ether as a solvent. In morepreferred embodiments the reaction is conducted at 0° C. Preferredsolvents for conducting the reaction include, but are not limited totetrahydrofuran (THF), tetrahydropyran, diethyl ether and the like.

3.2.2. Preparation of Compounds of Formula II

Compounds of formula II, which are substituted acetamides, may bepurchased or prepared from commercially available starting materials.Commercially available acetamides including: indole-3-acetamide,2-(5-methyl-1H-indol-3-yl)acetamide,2-(5-methoxy-1H-indol-3-yl)acetamide,2-(4-hydroxy-1H-indol-3-yl)acetamide, 2-phenylacetamide,2-(4-methylphenyl)acetamide, 4-hydroxyphenylacetamide,4-hydroxyphenylacetamide,N-cyclopentyl-2-(4-hydroxy-2-oxo-1,2-dihydro-3-quinolinyl)acetamide,2-phenoxyacetamide, 2-(2-methylphenoxy)acetamide,2-(4-fluorophenoxy)acetamide, 2-(4-pyridinyl)acetamide, and2-[(4-chlorophenyl)sulfanyl]acetamide are available from a variety ofsources including Sigma Aldrich Chemical Co., St. Louis Mo. A compoundof formula II may also be prepared from its corresponding free acid byconversion of the free acid to its acid chloride followed by reactionwith ammonia.

3.3. Additional Routes for the Preparation ofPyrroloquinolinyl-pyrrolidine-2,5-diones

In addition to those routes for the preparation ofpyrroloquinolinyl-pyrrolidine-2,5-diones described above, additionalroutes of preparing the compounds exemplified for(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneare described in Examples 62-64.

4. The Pharmaceutical Compositions and Formulations

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not so high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount,” as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is cancer. In another aspect, thedisease or condition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In one aspect, the active compounds are prepared with pharmaceuticallyacceptable carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 3000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

Preferably,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered at dosage of 360 mg, twice a day, for a maximal dailydosage of 720 mg.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis optionally administered to subjects or patients at an initial dosageof 10 mg twice daily for a maximal daily dose of 20 mg, with dosageescalation to administration of 360 mg twice daily for a maximal dailydosage of 720 mg. Preferred dosage forms of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneinclude, but are not limited to, caplets, tablets, pills, andfreeze-dried powder. For instance, a subject or patient is administeredone 360 mg caplet twice a day, or alternatively, two 180 mg caplets,twice a day, for a maximal daily dosage of 720 mg.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionecaplets or tablets are also formulated in 60 mg doses.

The pharmaceutical compositions can include co-formulations of any ofthe compounds described herein.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

EXAMPLES

Examples are provided below to further illustrate different features ofthe present invention. The examples also illustrate useful methodologyfor practicing the invention. These examples do not limit the claimedinvention.

Example 1 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dioneStep 1

To a solution of 1,2,3,4-tetrahydroquinoline (100 mL) in anhydroustetrahydrofuran (300 mL), bromoethylpyrruvate (53 mL) was added dropwiseover 30 minutes. The mixture was stirred for 24 hours at roomtemperature. The reaction mixture was filtered and the solid washed withtetrahydrofuran (100 mL). The filtrate was evaporated to dryness to give3-(3,4-dihydro-2H-quinolin-1-yl)-2-oxopropionic acid ethyl ester as abrown oil 117 g.

Step 2

Anhydrous magnesium chloride (29.4 g, 0.31 mol) was suspended in2-methoxyethanol (400 ml), and the mixture was stirred for 15 minutes at125° C. A solution of 3-(3,4-dihydro-2H-quinolin-1-yl)-2-oxopropionicacid ethyl ester (76.57 g 0.31 mol) in 2-methoxyethanol (100 ml) wasthen added and the mixture stirred at 125° C. for 60 minutes. Themixture was stirred for a further 5 hours at reflux, cooled andevaporated to dryness. The residue was then acidified with 2 Mhydrochloric acid (500 mL) and extracted with dichloromethane (3×500mL). The combined organic layers were then washed with 5% sodiumbicarbonate solution and dried over anhydrous magnesium sulfate beforebeing evaporated to dryness. The residue was then purified on a silicagel chromatography column, eluting with ethyl acetate/hexanes (1:4) toprovide 5,6-dihydro-4H-pyrrolo [3,2,1-ij]quinoline-1-carboxylic acidethyl ester (31.0 g, 47%). ¹H NMR (CDCl₃) 400 MHz δ: 7.9 (d, 1H, J=8Hz), 7.79 (s, 1H), 7.17 (m, 1H), 6.99 (d, 1H, J=7.2 Hz), 4.37 (m, 2H),4.18 (t, 2H, J=5.6 Hz), 3.0 (t, 2H, J=6 Hz), 2.24 (t, 2H, J=6 Hz), 1.42(t, 3H, J=7.2 Hz).

Step 3

To a solution of 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-1-carboxylicacid ethyl ester (31 g, 0.14 mol) in ethanol (200 mL) and water (200 mL)was added sodium hydroxide (30.8 g, 0.77 mol). The mixture was heated toreflux for 2 hours before being cooled to room temperature and dilutedwith water (2.64 L). The mixture was then washed with dichloromethane(2×300 mL) and the aqueous layer was acidified with concentratedhydrochloric acid to pH 1.0. The precipitate formed was collected byfiltration, washed with water and dried to yield5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-1-carboxylic acid as a darkyellow solid (23 g, 85%). ¹H NMR (DMSO-d₆) 400 MHz δ: 11.95 (brs, 1H),7.96 (s, 1H), 7.69 (d, 1H, J=8.4 Hz), 7.06 (t, 1H, J=6.8 Hz), 6.92 (d,1H, J=6.8 Hz), 4.19 (t, 2H, J=6 Hz), 2.91 (t, 2H, J=6 Hz), 2.11 (t, 2H,J=5.6 Hz).

Step 4

5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline-1-carboxylic acid (37.5 g,0.186 mol), copper chromite (13.5 g, 43 mmol) and quinoline (180 mL)were heated with stirring to 185° C. for 2 hours. The mixture wascooled, diluted with dichloromethane (1 L) and filtered over hyflo. Thefiltrate was washed with 2 M hydrochloric acid (2×600 mL) and twice with2 M sodium hydroxide (150 mL) before being evaporated to dryness. Theresidue was purified by silica gel chromatography, eluting with a ethylacetate/hexanes (1:6) to afford5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline (21 g, 72%) as a pale yellowsolid. ¹H NMR (CDCl₃) 400 MHz δ: 7.44 (dd, 1H, J=0.8 and 7.6 Hz), 7.07(d, 1H, J=3.2 Hz), 7.01 (t, 1H, J=7.2 Hz), 6.9 (dd, 1H, J=0.8 and 6.8Hz), 6.43 (d, 1H, J=3.2 Hz), 4.16 (t, 2H, J=6 Hz), 2.99 (t, 2H, J=6.4Hz), 2.24 (m, 2H).

Step 5

To a solution of 5,6-dihydro-4H-pyrroloquinoline (4.0 g, 25.3 mmol), inanhydrous ether (300 mL) at 0° C., was added oxalyl chloride (2.22 mL,25.3 mmol). The mixture was stirred for 30-45 minutes at 0° C. beforebeing cooled to −78° C. Sodium methoxide in methanol (0.5M) (60 mL) wasthen added slowly and the mixture allowed to warm to room temperature.The mixture was then diluted with ethyl acetate (200 mL), washed withwater (100 mL) followed by a wash with saturated aqueous sodium chloride(50 mL). The organic layer was dried over anhydrous sodium sulfate andevaporated to dryness. The residue was dissolved in ethyl acetate (100mL) filtered through a 2 inch plug of coarse silica gel and evaporatedto give 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoacetic acidmethyl ester as a yellow solid (5.3 g, 85%). ¹H NMR (CDCl₃) 400 MHz δ:8.3 (s, 1H), 8.14 (d, 1H), 7.22 (t, 1H), 7.04 (d, 1H), 4.2 (t, 2H), 3.95(s, 3H), 3.0 (t, 2H), 2.3 (t, 2H).

Step 6

To a solution of 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)oxoacetic acid methyl ester (1.0 g, 4.12 mmol) and indole-3-acetamide(0.8 g, 4.5 mmol) in anhydrous tetrahydrofuran at 0° C. was added asolution of potassium t-butoxide (1M in tetrahydrofuran) (12.4 mL, 12.4mmol) dropwise over 30 minutes. The mixture was stirred at 0° C. for 2hours. Concentrated hydrochloric acid (10 mL) was then added and themixture stirred for 1 hour at room temperature. The mixture was thendiluted with ethyl acetate (200 mL), washed twice with water (50 mL),and saturated aqueous sodium chloride solution (50 mL) and the organiclayer dried over anhydrous sodium sulfate. The residue was purified bysilica gel chromatography, eluting with ethyl acetate/hexanes (1:4) toafford3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dioneas a bright red solid (1.2 g, 80%). ¹H NMR (CDCl₃) 400 MHz δ: 8.5 (brs,1H), 7.78 (s, 1H), 7.63 (d, 1H, J=2.8 Hz), 7.44 (s, 1H), 7.35 (d, 1H,J=8 Hz), 7.16 (d, 1H, J=8.4 Hz), 7.11 (t, 1H, J=7.6 Hz), 6.86 (t, 1H,J=7.6 Hz), 6.80 (d, 1H, J=7.2 Hz), 6.64 (t, 1H, J=8 Hz), 6.57 (d, 1H,J=8 Hz), 4.2 (t, 2H, J=6 Hz), 2.96 (t, 2H, J=6 Hz), 2.24 (m, 2H).

Example 2 Preparation of(±)—Cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione Preparation of the(±)-cis compounds, (±)-trans compounds, or mixtures thereof wereobtained using reducing conditions as described in each of Procedures Athrough C Procedure A: Reduction of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dionewith Zn/Hg

The active zinc-mercury reducing agent for the reduction of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dionewas prepared from metallic zinc and HgCl₂. Zinc powder (2.5 g) andmercury (II) chloride (0.25 g) were suspended in de-ionized water (3 mL)and stirred for 20 minutes. A few drops of concentrated hydrochloricacid was then added and the mixture stirred for few minutes. The solidwas filtered off, washed with de-ionized water (50 mL), ethanol (50 mL)and dried.

To a suspension of the Zn(Hg) reducing agent prepared as above in dryethanol (50 mL) was added3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione(0.35 g, 95.4 μmol.). The mixture was heated to reflux for 30-60 minuteswhile dry hydrogen chloride gas was slowly passed through the mixture.The mixture was then cooled, filtered, and evaporated to dryness. A 5%potassium carbonate solution (150 mL) and ethyl acetate (300 mL) werethen added. The organic layer was dried over anhydrous magnesium sulfateand evaporated to give ˜2:1 mixture of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(0.2 g).

Procedure B: Reduction of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dionewith hydrogen in the presence of palladium on carbon

A suspension of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione(16 g, 43.6 mmol) and 10% palladium on carbon (Pd/C, wet catalyst) (8 g)were stirred under 1 atmosphere of hydrogen in methanol (600 mL) at roomtemperature for 48 hours. The catalyst was then filtered through a bedof Celite and the filtrate evaporated to dryness. The residue wasre-dissolved in methanol and the product precipitated by the addition ofcold water. The precipitate was filtered, washed with water and driedunder vacuum to yield(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(9.2 g). ¹H NMR (DMSO-d₆) 400 MHz δ: 11.56 (s, 1H), 10.66 (s, 1H), 7.43(d, 1H, J=7.6 Hz), 7.14 (d, 2H, J=8 Hz), 6.86-6.97 (m, 4H), 6.78 (t, 1H,J=7.2 Hz), 6.69 (d, 1H, J=6.8 Hz), 4.88 (dd, 2H, J=9.2 and 45.6 Hz),3.88 (m, 2H), 2.76 (t, 2H, J=5.6 Hz), 1.94 (t, 2H, J=6 Hz).

Procedure C: Reduction of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dioneby magnesium in methanol

Magnesium turnings (3.05 g, 0.125 mol) were added to a solution of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione(2.56 g, 6.97 mmol) in anhydrous methanol (100 mL) and heated to refluxunder an atmosphere of nitrogen for 40 minutes. After cooling to roomtemperature the mixture was poured into ethyl acetate (300 mL), washedwith 1M hydrochloric acid (300 mL) and water (500 mL). The organic layerwas dried over anhydrous sodium sulfate and evaporated to dryness. Theresidue was then purified by silica gel chromatography using 40-50%ethyl acetate in hexanes to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneas a pale pink solid (2.3 g). ¹H NMR (DMSO-d₆) 400 MHz δ: 11.54 (s, 1H),11.03 (s, 1H), 7.32-7.4 (m, 4H), 7.17 (d, 1H, J=7.2 Hz), 7.07 (t, 1H,J=7.6 Hz), 6.96 (t, 1H, J=7.6 Hz), 6.82-6.89 (m, 2H), 4.5 (dd, 2H, J=7.2and 20 Hz), 4.07 (t, 2H, J=5.2 Hz), 2.87 (t, 2H, J=6 Hz), 2.08 (m, 2H).

Example 3 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom(±)-Cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

A preparation of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(378 mg, 1.02 mmol) was heated to 50° C. in tert-butanol (10 mL) andpotassium t-butoxide (11 mg, 98 μmol) for 16 hours. The mixture waspoured into ethyl acetate (100 mL) and washed with water (100 mL). Theorganic layer was dried over anhydrous sodium sulfate and evaporated todryness to give(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneas a tan powder (276 mg). ¹H NMR (DMSO-d₆) 400 MHz δ: 11.54 (s, 1H),11.03 (s, 1H), 7.32-7.4 (m, 4H), 7.17 (d, 1H, J=7.2 Hz), 7.07 (t, 1H,J=7.6 Hz), 6.96 (t, 1H, J=7.6 Hz), 6.82-6.89 (m, 2H), 4.5 (dd, 2H, J=7.2and 20 Hz), 4.07 (t, 2H, J=5.2 Hz), 2.87 (t, 2H, J=6 Hz), 2.08 (m, 2H).

Example 4 Chromatographic Separation of3(R),4(S)-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand3(S),4(R)-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

A mixture of(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(135 mg) in methanol (10 mL) and acetonitrile (6 mL) was subjected topreparative supercritical fluid chromatography, using a chiral AD column20 mm×250 mm, eluting with 35% methanol/CO2 at a flow rate of 3.5mL/minutes. To give a faster eluting peak at 4.55 minutes (60 mg)assigned3(R),4(S)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a slower eluting peak 6.05 minutes (56 mg), assigned3(S),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.The absolute stereochemical assignments were based solely upon therelative retention time of related compounds and may be reversed.

Example 5 Chromatographic Separation of3(R),4(R)-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand3(S),4(S)-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

A mixture of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(200 mg) in acetonitrile (1 mL) was subjected to preparativesupercritical fluid chromatography using a CHIRALPAK® AD column (Daicel,U.S.A.) 20 mm×250 mm, eluting with 35% methanol/CO2 at a flow rate of3.5 mL/minutes. Chromatography yielded a faster eluting peak of thetrans isomer (82 mg) having a negative optical rotation assigned(−)-3(R),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand a slower eluting peak of the trans isomer (86 mg) having a positiveoptical rotation assigned(+)-3(S),4(R)-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Absolute stereochemical assignments were based solely upon relativeretention time of related compounds they may be reversed. All opticalrotation measurements were conducted in chloroform at 25° C. at 589 nm.

Crystals of the chromatographically separated (+) or (−) isomers oftrans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionemay be prepared from 2,2,2-trifluoroethanol using vapor stresstechniques and slow evaporation at 49° C. Crystals of these isomers mayalso be prepared from ethanol at room temperature by evaporationemploying seed crystals, such as those prepared by vapor stresstechniques.

Example 6 Preparation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-trifluoromethyl-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-trifluoromethyl-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2′-trifluoromethylphenyl acetamide in place of indole-3-acetamide instep 6. ¹H NMR (CDCl₃) 400 MHz δ: 8.16 (s, 1H), 7.83 (d, 2H, J=7.2 Hz),7.58 (m, 2H), 7.37 (d, 1H, J=7.6 Hz), 7.33 (s, 1H), 6.85 (d, 1H, J=6.8Hz), 6.66 (t, 1H, J=7.2 Hz), 5.96 (d, 1H, J=8.8 Hz), 4.2 (t, 2H, J=5.6Hz), 2.95 (t, 2H, J=6.4 Hz), 2.22 (m, 2H).

Example 7 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-thiophen-2-yl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-thiophen-2-yl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2-thienylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(CDCl₃) 400 MHz δ: 7.87 (s, 1H), 7.49 (d, 1H, J=5.2 Hz), 7.37 (s, 1H),7.3 (d, 1H, J=4 Hz), 7.02 (t, 1H, J=4 Hz), 6.89-6.98 (m, 2H), 6.53 (d,1H, J=7.6 Hz), 4.92 (t, 2H, J=6 Hz), 3.04 (t, 2H, J=6 Hz), 2.31 (m, 2H).

Example 8 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxy-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxy-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing3-methoxyphenylacetamide in place of indole-3-acetamide in step 6. ¹HNMR (CDCl₃) 400 MHz δ: 8.01 (s, 1H), 7.31 (s, 1H), 7.23 (t, 1H, J=7.6Hz), 7.09 (m, 2H), 6.87-6.92 (m, 2H), 6.73 (t, 1H, J=7.6 Hz), 6.14 (d,1H, J=8 Hz), 4.25 (t, 2H, J=5.2 Hz), 2.99 (t, 2H, J=5.6 Hz), 2.67 (m,2H).

Example 9 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-pyridin-2-yl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-pyridin-2-yl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employingpyridin-2-ylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(CDCl₃) 400 MHz δ: 8.58 (d, 1H, J=4.4 Hz), 8.12 (s, 1H), 7.78 (dt, 1H,J=1.6 and 7.6 Hz), 7.68 (d, 1H, J=8 Hz), 7.31 (s, 1H), 7.25 (m, 1H),6.87 (d, 1H, J=6 Hz), 6.68 (t, 1H, J=8 Hz), 5.91 (d, 1H, J=7.6 Hz), 4.24(t, 2H, J=5.6 Hz), 2.97 (t, 2H, J=6 Hz), 2.25 (m, 2H).

Example 10 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-methoxy-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-methoxy-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing4-methoxyphenylacetamide in place of indole-3-acetamide in step 6. ¹HNMR (CDCl₃) 400 MHz δ: 7.95 (s, 1H), 7.51 (m, 2H), 7.25 (s, 1H),6.85-6.89 (m, 3H), 6.75 (t, 1H, J=8 Hz), 6.24 (d, 1H, J=8 Hz), 4.26 (t,2H, J=5.6 Hz), 3.82 (s, 3H), 2.99 (t, 2H, J=6.4 Hz), 2.27 (m, 2H).

Example 11 Preparation of3-Benzo[1,3]dioxol-5-yl-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dione

3-Benzo[1,3]dioxol-5-yl-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing3,4-(methylenedioxy)phenylacetamide in place of indole-3-acetamide instep 6. ¹H NMR (CDCl₃) 400 MHz δ: 7.98 (s, 1H), 7.04-7.07 (m, 2H), 6.90(d, 1H, J=7.2 Hz), 6.76-6.82 (m, 2H), 6.30 (d, 1H, J=8 Hz), 5.98 (s,2H,), 4.26 (t, 2H, J=5.6 Hz), 2.99 (t, 2H, J=6 Hz), 2.28 (m, 2H).

Example 12 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-phenyl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-phenyl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employingphenylacetamide in place of indole-3-acetamide in step 6. ¹H NMR (CDCl₃)400 MHz δ: 8.01 (s, 1H), 7.52 (m, 2H), 7.35 (m, 3H), 7.27 (s, 1H), 6.87(d, 1H, J=7.2 Hz), 6.7 (t, 1H, J=7.2 Hz), 6.08 (d, 1H, J=8 Hz), 4.26 (t,2H, J=5.6 Hz), 2.99 (t, 2H, J=5.6 Hz), 2.27 (m, 2H).

Example 13 Preparation of3-Benzo[b]thiophen-2-yl-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dione

3-Benzo[b]thiophen-2-yl-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2-benzothiophenylacetamide in place of indole-3-acetamide in step 6. ¹HNMR (DMSO-d₆) 400 MHz δ: 11.11 (s, 1H), 8.14 (s, 1H), 8.01 (d, 1H, J=8Hz), 7.84 (s, 1H), 7.45 (d, 1H, J=8 Hz), 7.3 (t, 1H, J=7.2 Hz), 7.15 (t,1H, J=7.6 Hz), 6.71 (d, 1H, J=6.8 Hz), 6.43 (t, 1H, J=7.6 Hz), 5.99 (d,1H, J=8 Hz), 4.26 (t, 2H, J=5.2 Hz), 2.86 (t, 2H, J=5.6 Hz), 2.1 (m,2H).

Example 14 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-(3-phenoxy-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-phenoxy-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing3-phenoxyphenylacetamide in place of indole-3-acetamide in step 6. ¹HNMR (DMSO-d₆) 400 MHz δ: 11.03 (s, 1H), 8.01 (s, 1H), 7.43 (t, 1H, J=7.6Hz), 7.28 (d, 1H, J=7.6 Hz), 7.15 (t, 2H, J=7.6 Hz), 7.03 (t, 2H, J=7.6Hz), 6.92 (d, 1H, J=6.8 Hz), 6.8 (s, 1H), 6.76 (t, 1H, J=8 Hz), 6.60 (d,2H, J=7.6 Hz), 6.08 (d, 1H, J=8 Hz), 4.27 (t, 2H, J=5.6 Hz), 2.97 (t,2H, J=6 Hz), 2.16 (m, 2H).

Example 15 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-chloro-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-chloro-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing3-chlorophenylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.11 (s, 1H), 8.13 (s, 1H), 7.47-7.43 (m, 2H),7.36 (t, 1H, J=7.6 Hz), 7.29 (d, 1H, J=7.6 Hz), 6.86 (d, 1H, J=6.8 Hz),6.68 (t, 1H, J=7.6 Hz), 5.97 (d, 1H, J=8 Hz), 4.31 (t, 2H, J=5.6 Hz),2.93 (t, 2H, J=5.6 Hz), 2.16 (m, 2H).

Example 16 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-(2-chloro-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2-chlorophenylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.1 (s, 1H), 8.17 (s, 1H), 7.55 (d, 1H, J=8 Hz),7.45-7.49 (m, 1H), 7.36 (d, 2H, J=4.4 Hz), 6.81 (d, 1H, J=7.2 Hz), 6.58(t, 1H, J=8 Hz), 5.92 (d, 1H, J=8.4 Hz), 4.27 (m, 2H), 2.89 (t, 2H, J=6Hz), 2.11 (m, 2H).

Example 17 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,5-dimethoxy-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,5-dimethoxy-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2,5-dimethoxyphenylacetamide in place of indole-3-acetamide in step 6.¹H NMR (DMSO-d₆) 400 MHz δ: 10.93 (s, 1H), 8.06 (s, 1H), 6.97 (s, 2H),6.81 (d, 1H, J=7.6 Hz), 6.77 (s, 1H), 6.6 (t, 1H, J=8 Hz), 5.92 (d, 1H,J=8 Hz), 4.26 (t, 2H, J=5.2 Hz), 3.63 (s, 3H), 3.3 (s, 3H), 2.9 (t, 2H,J=5.6 Hz), 2.11 (m, 2H).

Example 18 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-4-fluoro-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-4-fluoro-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2-chloro-4-fluorophenylacetamide in place of indole-3-acetamide in step6. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.11 (s, 1H), 8.16 (s, 1H), 7.57 (dd,1H, J=2.8 and 9.2 Hz), 7.44 (dd, 1H, J=6.8 and 8.4 Hz), 7.28 (dt, 1H,J=2.4 and 8.4 Hz), 6.84 (d, 1H, J=7.2 Hz), 6.66 (t, 1H, J=8 Hz), 5.98(d, 1H, J=8 Hz), 4.27 (m, 2H), 2.9 (t, 2H, J=5.6 Hz), 2.11 (m, 2H).

Example 19 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-naphthalene-1-yl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-naphthalene-1-yl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing1-naphthylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.1 (s, 1H), 8.17 (s, 1H), 8.02 (d, 1H, J=8 Hz),7.97 (d, 1H, J=8 Hz), 7.75 (d, 1H, J=8 Hz), 7.43-7.55 (m, 3H), 7.37 (t,1H, J=8 Hz), 6.66 (d, 1H, J=6.8 Hz), 6.27 (t, 1H, J=8 Hz), 5.57 (d, 1H,J=8 Hz), 4.24 (t, 2H, J=5.2 Hz, 2.83 (t, 2H, J=5.6 Hz), 2.08 (m, 2H).

Example 20 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,6-dichloro-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,6-dichloro-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2,6-dichlorophenylacetamide in place of indole-3-acetamide in step 6. ¹HNMR (DMSO-d₆) 400 MHz δ: 11.23 (s, 1H), 8.27 (s, 1H), 7.53-7.62 (m, 3H),6.85 (d, 1H, J=7.2 Hz), 6.64 (t, 1H, J=8.4 Hz), 6.01 (d, 1H, J=8 Hz),4.27 (t, 2H, J=5.6 Hz), 2.9 (t, 2H, J=5.6 Hz), 2.11 (m, 2H).

Example 21 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-bromo-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-bromo-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2-bromophenylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.09 (s, 1H), 8.17 (s, 1H), 7.75 (m, 1H), 7.37 (m,2H), 7.33 (m, 1H), 6.81 (d, 1H, J=7.2 Hz), 6.58 (t, 1H, J=8 Hz), 5.95(d, 1H, J=8 Hz), 4.26 (t, 2H, J=5.6 Hz), 2.9 (t, 2H, J=5.6 Hz), 2.11 (m,2H).

Example 22 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-indol-1-yl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-indol-1-yl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employingN-indolyl-2-acetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.21 (s, 1H), 8.18 (s, 1H), 7.58 (d, 1H, J=8 Hz),7.52 (d, 1H, J=3.2 Hz), 7.01 (m, 2H), 6.91 (t, 1H, J=6.8 Hz), 6.74 (d,1H, J=2.8 Hz), 6.71 (d, 1H, J=7.2 Hz), 6.4 (t, 1H, J=8 Hz), 5.63 (d, 1H,J=8 Hz), 4.28 (t, 2H, J=4.8 Hz), 2.85 (t, 2H, J=5.6 Hz), 2.11 (m, 2H).

Example 23 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-pyridine-3-yl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-pyridine-3-yl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employingpyridine-3-ylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.14 (s, 1H), 8.53 (m, 2H), 8.12 (s, 1H), 7.78 (d,1H, J=7.6 Hz), 7.41 (dd, 1H, J=4.8 and 8 Hz), 6.86 (d, 1H, J=7.2 Hz),6.66 (t, 1H, J=7.6 Hz), 5.97 (d, 1H, J=8.4 Hz), 4.3 (t, 2H, J=5.2 Hz),2.93 (t, 2H, J=5.6 Hz), 2.16 (m, 2H).

Example 24 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-(5-bromo-1H-indol-3-yl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-bromo-1H-indol-3-yl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing5-bromo-1H-indoly-3-ylacetamide in place of indole-3-acetamide in step6. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.77 (s, 1H), 10.92 (s, 1H), 7.82 (s,1H), 7.69 (d, 1H, J=2.4 Hz), 7.33 (d, 1H, J=8.4 Hz), 7.10 (dd, 1H, J=2and 8.4 Hz), 6.99 (d, 1H, J=1.6 Hz), 6.76 (d, 1H, J=7.2 Hz), 6.55 (t,1H, J=8 Hz), 6.36 (d, 1H, J=8 Hz), 4.25 (t, 2H, J=5.6 Hz), 2.92 (t, 2H,J=5.6 Hz), 2.17 (m, 2H).

Example 25 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-pyridine-4-yl-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-pyridine-4-yl-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employingpyridine-4-ylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.17 (s, 1H), 8.53 (m, 2H), 8.54 (d, 2H, J=6 Hz),8.17 (s, 1H), 7.32 (d, 2H, J=4.8 Hz), 6.88 (d, 1H, J=7.2 Hz), 6.69 (t,1H, J=7.6 Hz), 5.93 (d, 1H, J=8 Hz), 4.31 (t, 2H, J=6 Hz), 2.94 (t, 2H,J=6 Hz), 2.16 (m, 2H).

Example 26 Preparation of 3-Biphenyl-4-yl-4-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-pyrrole-2,5-dione

3-Biphenyl-4-yl-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing4-phenylphenylacetamide in place of indole-3-acetamide in step 6. ¹H NMR(acetone-d₆) 400 MHz δ: 8.08 (s, 1H), 7.6-7.73 (m, 7H), 7.48 (t, 2H,J=6.8 Hz), 7.39 (d, 1H, J=7.2 Hz), 6.84 (d, 1H, J=8 Hz), 6.65 (t, 1H,J=8.4 Hz), 6.23 (t, 1H, J=7.2 Hz), 5.97 (d, 1H, J=8.4 Hz), 4.38 (m, 2H),2.98 (m, 2H), 2.28 (m, 2H).

Example 27 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-methanesulfonyl-phenyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-methanesulfonyl-phenyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing4-methanesulfonylphenylacetamide in place of indole-3-acetamide in step6. ¹H NMR (CDCl₃) 400 MHz δ: 8.09 (s, 1H), 7.9 (d, 2H, J=8.4 Hz), 7.71(d, 2H, J=8.4 Hz), 7.64 (s, 1H), 6.91 (d, 1H, J=7.2 Hz), 6.73 (t, 1H,J=7.6 Hz), 5.95 (d, 1H, J=8.4 Hz), 4.29 (t, 2H, J=5.6 Hz), 3.06 (s, 3H),3.0 (t, 2H, J=6 Hz), 2.29 (m, 2H).

Example 28 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-trifluoromethyl-quinolin-4-yl-sulfanyl)-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-trifluoromethyl-quinolin-4-yl-sulfanyl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing2-[[2-(trifluoromethyl)-4-quinolinyl]thio]acetamide in place ofindole-3-acetamide in step 6. ¹H NMR (CDCl₃) 400 MHz δ: 8.3 (d, 1H,J=7.6 Hz), 8.11 (d, 1H, J=8.4 Hz), 8.05 (s, 1H), 7.68-7.82 (m, 4H), 7.23(s, 1H), 6.83 (m, 2H), 4.21 (t, 2H, J=6 Hz), 2.92 (t, 2H, J=6 Hz), 2.21(m, 2H).

Example 29 Preparation of3-(4-Benzoyloxyphenyl)-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dione

3-(4-Benzoyloxyphenyl)-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing4-benzyloxyphenylacetamide in place of indole-3-acetamide in step 6. ¹HNMR (CDCl₃) 400 MHz δ: 7.95 (s, 1H), 7.5 (d, 2H, J=8.8 Hz), 7.33-7.43(m, 6H), 6.93 (d, 2H, J=8.8 Hz), 6.88 (d, 1H, J=7.2 Hz), 6.73 (t, 1H,J=7.2 Hz), 6.23 (d, 1H, J=8.4 Hz), 5.08 (s, 2H), 4.25 (t, 2H, J=5.6 Hz),2.99 (t, 2H, J=6 Hz), 2.27 (m, 2H).

Example 30 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-[4-(2-morpholin-4-yl-ethoxy)-phenyl]-pyrrole-2,5-dionewas prepared according to Example 1, steps 1-6, employing4-(2-morpholin-4-yl-ethoxy)-phenylacetamide in place ofindole-3-acetamide in step 6. ¹H NMR (CDCl₃) 400 MHz δ: 7.95 (s, 1H),7.48 (m, 3H), 6.86 (m, 3H), 6.74 (t, 1H, J=8 Hz), 6.23 (d, 1H, J=8. Hz),4.26 (t, 2H, J=5.2 Hz), 4.16 (t, 2H, J=5.6 Hz), 3.77 (t, 4H, J=4.8 Hz),2.99 (t, 2H, J=6 Hz), 2.87 (t, 2H, J=5.2 Hz), 2.65 (m, 4H), 2.28 (m,2H).

Example 31 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-1-naphthyl-1H-indol-3-yl)pyrrole-2,5-dione

A mixture of 1-naphthyl boronic acid (41 mg, 0.24 mmol),3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-bromo-1H-indol-3-yl)pyrrole-2,5-dione(88 mg, 0.2 mmol) (prepared as in Example 24),tetrakistriphenylphosphine palladium (5 mol %) in toluene (4 mL),ethanol (4 mL), saturated NaHCO₃ (1 mL), and water (2 mL) was heated at100° C. under nitrogen for 5 hours. After cooling to room temperature,the mixture was extracted with ethyl acetate (3×15 mL) and concentrated.The residue was purified by silica gel chromatography, eluting withethyl acetate/hexanes (1:4) to afford3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4(5-1-naphthyl-1H-indol-3-yl)pyrrole-2,5-dioneas a bright red solid (70 mg, 71%). ¹H NMR (CD₃OD) δ: 1.80-1.92 (m, 2H),2.72-2.80 (t, J=6.0 Hz, 2H), 3.94-3.99 (t, J=6.0 Hz, 2H), 6.50-6.58 (m,3H), 6.66 (s, 1H), 6.72 (m, 1H), 6.98 (dd, J=8.4 Hz, J′=2.0 Hz, 1H),7.00-7.50 (m, 2H), 7.28 (dd, J=6.8 Hz, F=8.4 Hz, 1H), 7.38-7.43 (m, 2H),7.61 (s, 1H), 7.72 (d, J=8.4 Hz, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.97 (s,1H).

Example 32 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-phenyl-1H-indol-3-yl)pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-phenyl-1H-indol-3-yl)pyrrole-2,5-dionewas prepared according to the method of Example 31 employing phenylboronic acid in place of 1-naphthyl boronic acid. ¹H NMR (CD₃OD) δ:2.10-2.18 (m, 2H), 2.90 (t, J=5.6 Hz, 2H), 4.18 (t, J=5.6 Hz, 2H), 6.63(t, J=7.6 Hz, 1H), 6.75-6.83 (m, 5H), 7.11-7.20 (m, 3H), 7.22 (dd, J=8.4Hz, F=1.2 Hz, 1H), 7.38 (d, J=8.4 Hz, 1H), 7.59 (s, 1H), 7.93 (s, 1H).

Example 33 Preparation of 3-(5,6-Dihydro-4H-pyrroloquinolin-1-yl)-4-(5-(4-methoxyphenyl)-1H-indol-3-yl)pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(4-methoxyphenyl)-1H-indol-3-yl)pyrrole-2,5-dionewas prepared according to the method of Example 31 employing4-methoxyphenyl boronic acid in place of 1-naphthyl boronic acid. ¹H NMR(CD₃OD) δ: 2.09-2.18 (m, 2H), 2.90 (t, J=6.0 Hz, 2H), 4.15 (t, J=6.0 Hz,2H), 6.62-6.68 (m, 2H), 6.73 (s, 4H), 6.77-6.82 (m, 2H), 7.18 (d, J=8.4Hz, 1H), 7.33 (d, J=8.4 Hz, 1H), 7.53 (s, 1H), 7.91 (s, 1H).

Example 34 Preparation of3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(3-methylphenyl)-1H-indol-3-yl)pyrrole-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(3-methylphenyl)-1H-indol-3-yl)pyrrole-2,5-dionewas prepared according to the method of Example 31 employing3-methylphenyl boronic acid in place of 1-naphthyl boronic acid. ¹H NMR(CD₃OD) δ: 2.00-2.10 (m, 2H), 2.11 (s, 3H), 2.81-2.88 (t, J=6.0 Hz, 2H),4.03-4.11 (t, J=5.6 Hz, 2H), 6.50 (d, J=7.2 Hz, 1H), 6.64 (t, J=7.6 Hz,1H), 6.74-6.81 (m, 3H), 6.86 (d, J=8.0 Hz, 1H), 6.94 (d, J=7.6 Hz, 1H),7.03 (t, J=7.2 Hz, 1H), 7.22 (dd, J=8.4 Hz, J′=2.0 Hz, 1H), 7.36 (d,J=8.8 Hz, 1H), 7.48 (s, 1H), 7.90 (s, 1H).

Example 35 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione

3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-bromo-1H-indol-3-yl)pyrrole-2,5-dione,prepared as in Example 24, was reduced with Mg in methanol as describedin Example 2, Procedure C, to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-bromo-1H-indol-3-yl)pyrrolidine-2,5-dione.¹H NMR (CD₃OD) δ: 2.18-2.26 (m, 2H), 2.96 (t, J=6.0 Hz, 2H), 4.12 (t,J=6.4 Hz, 2H), 4.40 (d, J=6.8 Hz, 1H), 4.52 (d, J=6.8 Hz, 1H), 6.86-6.96(m, 2H), 7.08 (s, 1H), 7.13-7.30 (m, 5H)

Example 36 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(5-phenyl-1H-indol-3-yl)pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-phenyl-1H-indol-3-yl)pyrrole-2,5-dione,prepared as in Example 32, was reduced with Mg in methanol as describedin Example 2, Procedure C, to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-phenyl-1H-indol-3-yl)pyrrolidine-2,5-dione.¹H NMR (CD₃OD) δ: 2.00-2.16 (m, 2H), 2.94 (t, J=6.0 Hz, 2H), 3.92-3.99(m, 1H), 4.00-4.08 (m, 1H), 4.36 (d, J=6.4 Hz, 1H), 4.68 (d, J=6.4 Hz1H), 6.88-6.97 (m, 2H), 7.04 (s, 1H), 7.12-7.15 (m, 1H), 7.17-7.47 (m,9H).

Example 37 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(1-naphthyl)-1H-indol-3-yl)pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-1-naphthyl-1H-indol-3-yl)pyrrole-2,5-dione,prepared as in Example 31, was reduced with Mg in methanol as describedin Example 2, Procedure C, to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(1-naphthyl)-1H-indol-3-yl)pyrrolidine-2,5-dione.¹H NMR (CD₃OD) δ: 1.85-1.95 (m, 1H), 1.95-2.05 (m, 1H), 2.74-2.88 (m,2H), 3.72-3.83 (m, 1H), 3.88-3.98 (m, 1H), 4.40 (d, J=6.4 Hz, 1H), 4.62(d, J=6.4 Hz, 1H), 6.80 (d, J=6.8 Hz, 1H), 6.78 (t, J=8.0 Hz, 1H), 7.46(s, 1H), 7.07-7.13 (m, 2H), 7.18-7.23 (dd, J=8.4 Hz, J=1.6 Hz, 2H),7.27-7.34 (m, 2H), 7.41-7.49 (m, 3H), 7.78-7.83 (dd, J=8.4 Hz, J=3.2 Hz,2H), 7.86-7.90 (d, J=7.6 Hz, 1H).

Example 38 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(4-methoxyphenyl)-1H-indol-3-yl)pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(4-methoxyphenyl)-1H-indol-3-yl)pyrrole-2,5-dione,prepared as in Example 33, was reduced with Mg in methanol as describedin Example 2, Procedure C, to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(4-methoxyphenyl)-1H-indol-3-yl)pyrrolidine-2,5-dione.¹H NMR (CD₃OD) δ: 2.03-2.22 (m, 2H), 2.98 (t, J=6.0 Hz, 2H), 3.80 (s,3H), 3.97-4.06 (m, 1H), 4.06-4.14 (m, 1H), 4.38 (d, J=6.8 Hz, 1H), 4.67(d, J=6.8 Hz, 1H), 6.86 (d, J=8.4 Hz, 2H), 6.91-7.00 (m, 2H), 7.08 (s,2H), 7.17-7.27 (m, 4H), 7.31 (d, J=8.4 Hz, 1H), 7.37 (d, J=8.8 Hz, 1H).

Example 39(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(3-methylphenyl)-1H-indol-3-yl)pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(3-methylphenyl)-1H-indol-3-yl)pyrrole-2,5-dione,prepared as in Example 34, was reduced with Mg in methanol as describedin Example 2, Procedure C, to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(5-(3-methylphenyl)-1H-indol-3-yl)pyrrolidine-2,5-dione.¹H NMR (CD₃OD) δ: 1.98-2.18 (m, 2H), 2.34 (s, 3H), 2.85-3.00 (m, 2H),3.90-3.98 (m, 1H), 3.98-4.09 (m, 1H), 4.35 (d, J=7.2 Hz, 1H), 4.64 (d,J=6.8 Hz, 1H), 6.88-6.99 (m, 2H), 7.00-7.10 (m, 3H), 7.13-7.26 (m, 5H),7.36 (m, 2H).

Example 40 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-4-fluorophenyl)pyrrolidine-2,5-dione

To a solution of 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)oxoacetic acid methyl ester (0.243 g, 1 mmol) and2-chloro-4-fluorophenylacetamide (1 mmol) in anhydrous tetrahydrofuran(5 mL) at 0° C. was added a solution of potassium t-butoxide (1 M intetrahydrofuran) (2.5 mL, 2.5 mmol). The mixture was stirred at 0° C.for 2 hours. Concentrated hydrochloric acid (0.5 mL) was then added andthe mixture stirred for 1 hour at room temperature. The mixture was thendiluted with ethyl acetate (20 mL), washed with water (2×15 mL) andsaturated aqueous sodium chloride solution (15 mL). The organic layerwas then dried over anhydrous sodium sulfate and concentrated underreduced pressure to yield an oil. This residue was diluted in anhydrousmethanol (15 mL) and the resulting solution charged with oven driedmagnesium turnings (0.5 g, 20.5 mmol) and stirred at 70° C. in aventilated vial until the Mg turnings fully dissolved or for two hours.The vial was then allowed to cool to room temperature. The mixture wasdiluted with ethyl acetate (25 mL) and washed with 10% hydrochloric acid(2×25 mL) and saturated aqueous sodium chloride solution (20 mL). Theorganic layer was dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gelchromatography, eluting with an ethyl acetate/hexanes gradient (10%ethyl acetate to 50% ethyl acetate over 40 minutes) to yield (25.6 mg,6.7%) of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-4-fluorophenyl)pyrrolidine-2,5-dione.¹H NMR (DMSO-d₆) 400 MHz δ: 12.5 (s, 1H), 7.52 (t, 1H, J=6.4 Hz), 7.49(dd, 1H, J=6.4 2.4 Hz), 7.34 (s, 1H), 7.21 (td, 1H, J=6.0 2.8 Hz), 7.10(d, 1H, J=7.6 Hz), 6.87 (m, 2H), 4.67 (d, 1H, J=8.0 Hz), 4.51 (d, 1H,J=7.2 Hz), 2.90 (t, 2H, J=5.6 Hz), 2.11 (t, 2H, J=5.2 Hz).

Example 41 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,6-dichlorophenyl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,6-dichlorophenyl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 2,6-dichlorophenylacetamide. Yield52.2 mg, 13.0%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.82 (s, 1H), 7.34 (m, 3H),7.10 (d, 1H, J=7.2 Hz), 6.87 (m, 2H), 5.16 (d, 1H J=7.6 Hz), 5.10 (d,1H, J=7.6 Hz), 2.91 (t, 2H, J=6.0 Hz) 2.10 (m, 2H).

Example 42 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(4-bromophenyl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-bromophenyl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 4-bromophenylacetamide. Yield 33.1mg, 8.1%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.55 (s, 1H), 7.53 (dt, 2H, J=8.82.0 Hz), 7.34 (dt, 3H, J=8.0 2.0 Hz), 7.15 (dd, 1H, J=7.6 1.0 Hz), 6.86(m, 2H), 4.53 (d, 1H, J=8.0 Hz), 4.37 (d, 1H, J=8.0 Hz), 4.10 (t, 2H,J=1.6 Hz), 2.90 (t, 2H, J=2.0 Hz), 2.12 (t, 2H, J=1.8 Hz).

Example 43 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(4-chlorophenyl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-chlorophenyl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 4-chlorophenylacetamide. Yield32.7 mg, 9.0%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.54 (s, 1H), 7.40 (m, 4H),7.33 (s, 1H), 7.15 (dd, 1H, J=6.8 0.8 Hz), 6.86 (m, 2H), 4.54 (d, 1H,J=8.0 Hz), 7.38 (d, 1H, J=7.6 Hz), 4.10 (t, 2H, J=5.6 Hz), 2.90 (t, 2H,J=6.0 Hz), 2.11 (m, 2H).

Example 44 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-trifluoromethoxyphenyl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-trifluoromethoxyphenyl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 4-trifluoromethoxyphenylacetamide.Yield 67.8 mg, 16.4%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.56 (s, 1H), 7.52(d, 2H, J=8.4 Hz), 7.35 (s, 1H), 7.33 (d, 2H, J=8.0 Hz), 7.15 (d, 1H,J=7.2 Hz), 6.86 (m, 2H), 4.58 (d, 1H, J=8.0 Hz), 4.45 (d, 1H, J=8.0 Hz),4.10 (t, 2H, J=6.0 Hz), 2.90 (t, 2H, J=6.0), 2.10 (t, 2H, J=5.6).

Example 45 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(thiophen-3-yl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(thiophen-3-yl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with thiophen-3-ylacetamide. Yield 50.3mg, 15.0%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.50 (s, 1H), 7.52 (m, 1H), 7.49(m, 1H), 7.35 (s, 1H), 7.21 (dd, 1H, J=4.0 1.2 Hz), 7.16 (d, 1H, 7.6Hz), 6.89 (d, 1H, J=4.4 Hz), 6.85 (t, 1H, J=6.8 Hz), 4.56 (d, 1H, J=7.2Hz), 4.41 (d, 1H, J=7.2 Hz), 4.10 (t, 2H, J=6.0 Hz), 2.90 (t, 2H, J=6.0Hz), 2.10 (m, 2H).

Example 46 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-fluorophenyl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-fluorophenyl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 2-fluorophenylacetamide. Yield30.6 mg, 8.8%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.64 (s, 1H), 7.36 (m, 3H),7.17 (m, 3H), 6.84 (m, 2H), 4.44 (d, 1H, J=7.2 Hz), 4.40 (d, 1H, J=7.6Hz), 4.10 (s, 2H), 2.88 (s, 2H), 2.09 (s, 2H).

Example 47 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(2-thiophen-2-yl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-thiophen-2-yl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 2-thiophen-2-ylacetamide. Yield30.6 mg, 8.8%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.58 (s, 1H), 7.45 (dd, 1H,J=5.2 0.8 Hz), 7.40 (s, 1H), 7.22 (d, 1H, J=8.0 Hz), 7.12 (d, 1H, J=3.2Hz), 6.99 (dd, 1H, J=5.2 and 3.6 Hz), 4.63 (d, 1H, J=8.0 Hz), 4.60 (d,1H, J=7.6 Hz), 2.90 (t, 2H, J=6.0 Hz), 2.12 (t, 2H, J=6.0 Hz).

Example 48 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,4-dichlorophenyl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,4-dichlorophenyl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 2,4-dichlorophenylacetamide. Yield20.9 mg, 5.2%. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.65 (s, 1H), 7.69 (s, 1H),7.51 (d, 1H, J=8.0 Hz), 7.43 (d, 1H, J=8.0 Hz), 7.34 (s, 1H), 7.12 (m,1H), 6.87 (m, 2H), 4.65 (d, 1H, J=7.6 Hz), 4.55 (d, 1H, J=7.6 Hz), 4.10(t, 2H, J=6.0 Hz), 2.90 (t, 2H, J=6.0), 2.12 (t, 2H, J=6.0 Hz).

Example 49 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-phenyl-pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-phenyl-pyrrolidine-2,5-dionewas prepared according to the Example 40 replacing2-chloro-4-fluorophenylacetamide with phenylacetamide. ¹H NMR (DMSO-d₆)400 MHz δ: 11.511 (s, 1H), 7.24-7.36 (m, 6H), 7.13 (d, 1H, J=7.2),6.8-6.88 (m, 2H), 4.49 (d, 1H, J=8.0 Hz), 4.3 (d, 1H, J=7.6 Hz), 4.08(m, 2H), 2.88 (m, 2H), 2.088 (m, 2H).

Example 50 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chlorophenyl)-pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chlorophenyl)-pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with 2-chlorophenylacetamide. ¹H NMR(DMSO-d₆) 400 MHz δ: 11.655 (s, 1H), 7.41-7.48 (m, 2H), 7.27-7.35 (m,3H, J=7.2), 7.87 (d, 1H, J=7.6), 6.81-6.88 (m, 2H), 4.632 (d, 1H, J=7.6Hz), 4.494 (d, 1H, J=7.2), 4.07-4.10 (m, 2H), 2.884 (m, 2H), 2.09 (m,2H).

Example 51 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(N-methylindol-3-yl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(N-methylindol-3-yl)pyrrolidine-2,5-dionewas prepared according to Example 40 replacing2-chloro-4-fluorophenylacetamide with N-methylindol-3-ylacetamide. ¹HNMR (DMSO-d₆) 400 MHz δ: 11.55 (s, 1H), 7.44-7.34 (m, 4H), 7.2-7.18 (m,2H), 7.01 (t, 1H), 6.82-6.89 (m, 2H), 4.49 (dd, 2H), 4.093 (t, 2H),4.093 (t, 2H), 3.73 (s, 3H), 2.89 (t, 2H), 2.07 (m, 2H).

Example 52 Preparation of(±)—Cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-methoxyphenyl)-pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4(4-methoxy-phenyl)-pyrrole-2,5-dione,prepared as in Example 10 and was reduced by employing the method ofExample 2, Protocol B, to yield(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-methoxyphenyl)-pyrrolidine-2,5-dione.¹H NMR (CDCl₃) 400 MHz δ: 8.62 (s, 1H), 7.15 (d, 1H, J=7.6 Hz), 6.8-6.93(m, 4H), 6.7 (s, 1H), 6.55 (d, 2H, J=8.4 Hz), 4.8 (d, 1H, J=8.8 Hz),4.48 (d, 1H, J=8.8 Hz), 3.96 (m, 2H), 3.63 (s, 3H), 2.87 (t, 2H, J=6Hz), 2.10 (m, 2H).

Example 53 Preparation of (±)—Cis-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(2,5-dimethoxyphenyl)-pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,5-dimethoxy-phenyl)-pyrrole-2,5-dione,prepared as in Example 17, was reduced by employing the method ofExample 2, Protocol B, to yield(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2,5-dimethoxyphenyl)-pyrrolidine-2,5-dione.¹H NMR (CDCl₃) 400 MHz δ: 8.0 (s, 1H), 7.19 (d, 1H, J=7.6 Hz), 6.89 (t,1H, J=7.2 Hz), 6.77 (d, 2H, J=7.2 Hz), 6.44-6.51 (m, 3H), 4.84 (d, 2H,J=9.6 Hz), 3.88-4.00 (m, 2H), 3.6 (s, 3H), 3.49 (s, 3H), 2.8 (m, 2H),2.05 (m, 2H).

Example 54 Preparation of (±)—Cis-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(2-chloro-4-fluoro-phenyl)-pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-4-fluoro-phenyl)-pyrrole-2,5-dione,prepared as in Example 18, was reduced by employing the method ofExample 2, Protocol B, to yield(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(2-chloro-4-fluoro-phenyl)-pyrrolidine-2,5-dione.¹H NMR (DMSO-d₆) 400 MHz δ: 11.82 (s, 1H), 7.02-7.18 (m, 4H), 6.7-6.85(m, 3H), 5.01 (d, 1H, J=9.2 Hz), 4.79 (d, 2H, J=9.6 Hz), 3.96 (m, 2H),2.79 (m, 2H), 1.97 (m, 2H).

Example 55 Preparation of (±)—Cis-3-(5,6-dihydro-4H-pyrroloquinolin-1-yl)-4-(3-chlorophenyl)-pyrrolidine-2,5-dione

3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-chloro-phenyl)-pyrrole-2,5-dione,prepared as in Example 15, was reduced by employing the method ofExample 2, Procedure B, to yield(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-chlorophenyl)-pyrrolidine-2,5-dione.¹H NMR (DMSO-d₆) 400 MHz δ: 11.66 (s, 1H), 7.13 (d, 1H, J=8 Hz),6.95-7.02 (m, 5H), 6.78 (t, 1H, J=7.6 Hz), 6.7 (d, 1H, J=7.2 Hz), 4.84(d, 1H, J=9.2 Hz), 4.65 (d, 2H, J=8.8 Hz), 3.9-4.03 (m, 2H), 2.79 (t,2H, J=5.6 Hz), 1.97 (m, 2H).

Example 56 Preparation of (±)-Phosphoric acidmono-[trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethyl]esterStep 1

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(3.0 g, 8.13 mmol, prepared as in Example 2, Procedure C) andformaldehyde (30 mL, 37% in water) in tetrahydrofuran (30 mL) werestirred for 14-16 hours at room temperature. The mixture was then takenup in ethyl acetate (50 mL) and water (50 mL). The organic layer waswashed with brine and dried over sodium sulfate. Solvent was removedunder reduced pressure and residue was purified using a silica gelchromatography column eluted with EtOAc/Hexane 1:1 to yield 2.5 g, 77%,of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dionean orange foamy solid (2.5 g, 77%).

Step 2

(±)-Trans-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione(0.06 g) in anhydrous tetrahydrofuran (5 mL) was treated withdibenzylphosphoramidate (0.156 mL, 3.5 equivalents) followed by theaddition of tetrazole (3% solution in acetonitrile, 2 mL). The reactionmixture was stirred at room temperature for 20 min and cooled to −78° C.A solution of m-chloroperbenzoic acid (70%, 0.162 g) in dichloromethane(2 mL) was added at −78° C. After 5 min at −78° C., the reaction wasbrought to room temperature and stirred for 5 min. Solvents were removedunder reduced pressure and the residue was purified by flashchromatography on a silica column, eluted with ethyl acetate, hexane togive phosphoric acid dibenzyl estertrans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethylester as a solid (70 mg). ¹H NMR (DMSO-d₆) 400 MHz δ: 11.10 (s, 1H),7.32-7.39 (m, 12H), 6.84-7.24 (m, 2H), 5.49 (brs, 2H), 5.03 (m, 4H),4.61 (dd, 2H), 4.06 (brs, 2H), 2.87 (brs, 2H), 2.07 (brs, 2H).

Step 3

The phosphoric acid dibenzyl ester of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-yl-methylester (0.160 g) in methanol (2 mL) and Pd/C (10%, 20 mg) was stirred atroom temperature under 1 atmosphere of hydrogen for two hours. Themixture was filtered over Celite and the solvent removed to give(±)-phosphoric acidmono-[trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethyl]ester(0.110 g).

Example 57 Preparation of (±)-trans-2-Amino-propionicacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethyl ester Step 1

To a solution of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione(0.5 mmol) in tetrahydrofuran (8 mL) was added N-carbobenzyloxy alanine(1.1 equivalents) followed by the addition of HBTU (1.5 equivalents) andDIPEA (2.2 equivalents). The mixture was stirred at room temperature for15 h. The solvents were removed under reduced pressure and the residuewas taken up in ethyl acetate and water (1:1, 15 mL). The organic layerwas separated and dried. The residue was purified by silica gelchromatography to provide the N-carbobenzyloxy protected product.

Step 2

A solution of the N-carbobenzyloxy protected product from Step 1 (0.5mmol) in methanol (8 mL) and a few drops of 4 M HCl in ethyl acetate and10% Pd/C (10% w/w) were stirred at room temperature under 1 atmosphereof hydrogen for 2 hours. The mixture was then filtered over celite andthe solvent removed to provide final product (±)-trans-2-amino-propionicacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethylester. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.10 (s, 1H), 8.57 (s, 2H),6.84-7.41 (m, 9H), 5.61 (m, 2H), 4.62 (dd, 2H), 4.07 (brs, 2H), 3.72(brm, 1H), 2.87 (brs, 2H), 2.23 (s, 6H), 2.08 (brs, 2H), 1.40 (d, J=6.4Hz, 3H).

Example 58 Preparation of (±)-trans-2-Amino-aceticacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethyl ester

(±)-trans-2-Amino-aceticacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethylester was prepared as in Example 57 by replacing N-carbobenzyloxyalanine with N-carbobenzyloxy glycine. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.19(s, 1H), 8.46 (s, 2H), 6.82-7.43 (m, 9H), 5.61 (s, 2H), 4.65 (dd, 2H),4.08 (brt, J=5.6 Hz, 2H), 3.88 (brs, 2H), 2.87 (t, J=5.6 Hz, 2H), 2.48(s, 2H), 2.08 (t, J=4.8 Hz, 2H).

Example 59 Preparation of (±)-trans-2-dimethylamino-aceticacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethylester

To a solution of(±)-trans-3-(5,6-Dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1-hydroxymethyl-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione(0.5 mmol) in tetrahydrofuran (8 mL) was added N,N-dimethylglycine (1.1equivalents) followed by the addition of HBTU (1.5 equivalents) andDIPEA (N,N-diisopropylethylamine, 2.2 equivalents). The mixture wasstirred at room temperature for 15 hours. The solvents were removedunder reduced pressure and the residue was taken up in ethyl acetate andwater (1:1, 15 mL). The organic layer was separated and dried to yield aresidue. The residue was purified by chromatography on a silica gelcolumn eluted with ethyl acetate hexanes to yield(±)-trans-2-dimethylamino-aceticacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethyl ester. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.10 (s, 1H), 6.82-7.41 (m,9H), 5.70 (m, 2H), 4.62 (dd, 2H), 4.07 (brs, 2H), 3.23 (s, 2H), 2.87(brs, 2H), 2.23 (s, 6H), 2.08 (brs, 2H).

Example 60 Preparation of (±)-trans-Isonicotinicacid-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethylester

(±)-trans-Isonicotinic acid3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-1-ylmethylester was prepared as in Example 59 by replacing N,N-dimethylglycinewith 4-carboxypyridine. ¹H NMR (DMSO-d₆) 400 MHz δ: 11.19 (s, 1H), 8.83(d, 2H), 7.83 (d, 2H), 6.83-7.42 (m, 9H), 5.88 (s, 2H), 4.65 (dd, 2H),4.05 (brt, 2H), 2.86 (brs, 2H), 2.08 (brs, 2H).

Example 61 Preparation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrole-2,5-dioneand(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrolidine-2,5-dione

Step 1: To a solution of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrole-2,5-dione(100 mg, see Example 1) in anhydrous dimethylformamide (5 mL) was addedpotassium carbonate (0.5 g) and methyl iodide (0.1 mL). The mixture wasstirred at room temperature for 48 hours then poured into ethyl acetate(100 mL), washed with water (100 mL), dried over anhydrous sodiumsulfate and evaporated to give3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrole-2,5-dioneas a red solid (93 mg).

Step 2: To a solution of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrole-2,5-dione(93 mg) in methanol (5 mL) and ethylacetate (5 mL) was added 10% Pd—C(50 mg) and the mixture stirred at room temperature under 1 atmosphereof hydrogen for 48 hours. Toluene (50 mL) was added and the mixtureagain stirred at room temperature under 1 atmosphere of hydrogen for 2hours. The mixture was then filtered through a pad of celite andevaporated to dryness to yield a residue. The residue was purified usingsilica gel chromatography eluting with 35-40% ethylacetate in hexanes togive(±)-cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1-methylindol-3-yl)-1-methylpyrrolidine-2,5-dioneas a pale yellow solid (53 mg). ¹H NMR (CDCl₃) 400 MHz δ: 7.23 (s, 1H),7.05-7.07 (m, 2H), 7.01 (d, 1H, J=7.2 Hz), 6.92-6.97 (m, 1H), 6.85 (t,1H, J=7.2 Hz), 6.74 (d, 1H, J=6.8 Hz), 6.64 (d, 2H, J=6.4 Hz), 4.78 (m,2H), 3.75-3.84 (m, 2H), 3.45 (s, 3H), 3.27 (s, 3H), 2.79 (t, 2H, J=5.6Hz), 1.98 (m, 2H).

Example 62 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionemay be prepared by reacting 1H-indole and3,4-dibromo-1-phenyl-pyrrole-2,5-dione in the presence of methylmagnesium bromide to yield3-bromo-4-(1H-indol-3-yl)-1-phenyl-pyrrole-2,5-dione. The3-bromo-4-(1H-indol-3-yl)-1-phenyl-pyrrole-2,5-dione is subsequentlyreacted with 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinoline and LiHMDS(lithium hexamethyldisilane) in toluene or(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-boranediol and Pd(PPh₃)₄(tetrakis(triphenylphosphine)palladium) to yield3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-1-phenyl-pyrrole-2,5-dione,which is reduced and deprotected by treatment with Mg in methanol, as inExample 2 procedure C, followed by catalytic hydrogenation overpalladium on carbon to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.Bnz is benzyl.

Example 63 Preparation of (±)-Trans-3-(5,6-dihydro-4H-pyrrolopyrrolidine-2,5-dione

(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionemay be prepared by reacting 1-allyl-7-bromo-1H-indole with (COCl)₂(oxalyl chloride) and sodium methoxide in a polar aprotic solvent suchas dichloromethane to yield (1-allyl-7-bromo-1H-indol-3-yl)-oxo-aceticacid methyl ester, which is subsequently reacted with2-(1H-indol-3-yl)-acetamide and tBuOK (potassium tert-butoxide) in THFto yield3-(1-allyl-7-bromo-1H-indol-3-yl)-4-(1H-indol-3-yl)-pyrrole-2,5-dione.Reduction of the3-(1-allyl-7-bromo-1H-indol-3-yl)-4-(1H-indol-3-yl)-pyrrole-2,5-dione byMg in refluxing methanol, as in Example 2 procedure C, yields3-(1-allyl-7-bromo-1H-indol-3-yl)-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione,which is treated with 9-BBN (9-borabicyclo[3.3.1]nonane) and Pd(PPh₃)₄(tetrakis(triphenylphosphine)palladium) to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Example 64 Preparation of(±)—Cis-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione

The cis and trans isomers of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionemay be prepared beginning with the reaction of(1H-indol-3-yl)-oxo-acetic acid methyl ester and(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-acetic acid methyl esterin the presence of a base such as LDA (lithium diisopropylamide) in apolar aprotic solvent such as THF to yield2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-but-2-enedioicacid dimethyl ester. Alternatively,2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-but-2-enedioicacid dimethyl ester may be prepared by reaction of(1H-indol-3-yl)-acetic acid methyl ester and(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-oxo-acetic acid methylester in the presence of a base (e.g., LDA) in THF. The2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-but-2-enedioicacid dimethyl ester is reduced by catalytic hydrogenation over a noblemetal catalyst (e.g., Pd on charcoal) to give2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-succinicacid dimethyl ester, which is reacted with benzylamine (PhCH₂NH₂) toyield a mixture of cis and trans3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-1-phenyl-pyrrolidine-2,5-dione.The mixture of cis and trans isomers may be deprotected by catalytichydrogenation over Pd on charcoal (Pd—C) to give rise to a mixture ofcis and trans3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione.The cis and trans isomers may be separated to give all four cis andtrans isomers (e.g., by chromatography as in Examples 4 and 5). Thedeprotected mixture of cis and trans isomers may be treated withpotassium tert-butoxide in tert-butanol (as in Example 3) or a mixtureof THF and tert-butanol at 50° C. to yield a mixture with a predominanceof the trans isomers. Alternatively, the2-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-3-(1H-indol-3-yl)-succinicacid dimethyl ester can be reacted with ammonia in methanol at elevatedtemperatures to yield predominantly the cis isomers of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-pyrrolidine-2,5-dione,which may be isomerized to yield predominately the trans isomers of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)-pyrrolidine-2,5-dionewith potassium tert-butoxide in tert-butanol (as in Example 3) or amixture of THF and tert-butanol at 50° C.

Example 65 Preparation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxyphenyl)-1H-pyrrole-2,5-dione

To a mixture of 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl) oxoaceticacid methyl ester (0.50 g, 2.05 mmol) and 2-(3-methoxyphenyl)acetamide(0.37 g, 2.26 mmol) in anhydrous tetrahydrofuran (5 mL) was addedpotassium tert-butoxide (1.0 M in tetrahydrofuran, 6.17 mL, 6.17 mmol)dropwise at 0° C. The mixture was stirred at 0° C. for 3 hours thenconcentrated hydrochloric acid (1.5 mL) was added at 0° C. The resultingmixture was stirred for 1 hour, diluted with ethyl acetate (150 mL),washed with water (2×50 mL), dried over anhydrous sodium sulfate andconcentrated to give 0.91 g of an orange solid. The residue was purifiedby column chromatography eluting with 20-40% ethyl acetate in hexane togive3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxyphenyl)-1H-pyrrole-2,5-dione.Mp 99-101° C.; ¹H NMR (CDCl₃) 400 MHz δ: 8.01 (s, 1H), 7.81 (bs, 1H),7.20-7.25 (m, 1H), 7.06-7.08 (m, 2H), 6.85-6.91 (m, 2H), 7.25 (t, 1H),6.13 (d, J=8.0 Hz, 1H), 4.24 (t, 2H), 3.66 (s, 3H), 2.97 (t, J=6.0 Hz,2H), 2.22-2.26 (m, 2H).

Example 66 Preparation of3-[4-(benzyloxy)phenyl]-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1H-pyrrole-2,5-dione

3-[4-(benzyloxy)phenyl]-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1H-pyrrole-2,5-dionewas prepared according to Example 65, employing2-(4-(benzyloxy)phenyl)acetamide in place of2-(3-methoxyphenyl)acetamide. Mp 262-265° C.; ¹H NMR (DMSO-d₆) 400 MHzδ: 10.96 (s. 1H), 8.01 (d, 1H), 7.33-7.45 (m, 7H), 6.99 (d, J=6.8 Hz,2H), 6.83 (d, J=7.2 Hz, 1H), 6.63 (t, 1H), 6.09 (d, J=8.4 Hz, 1H), 5.13(s, 2H), 4.27 (m, 2H), 2.92 (m, 2H), 2.15 (m, 2H).

Example 67 Preparation of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-fluorophenyl)-1H-pyrrole-2,5-dione

3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-fluorophenyl)-1H-pyrrole-2,5-dionewas prepared according to Example 65, employing2-(4-fluorophenyl)acetamide in place of 2-(3-methoxyphenyl)acetamide. Mp234-235° C.; ¹H NMR (DMSO-d₆) 400 MHz δ: 11.05 (s. 1H), 8.07 (d, 1H),7.42-7.46 (m, 2H), 7.71-7.22 (m, 2H), 6.83 (d, J=7.2 Hz, 1H), 6.65-6.69(m, 1H), 6.00 (d, J=8.0 Hz, 1H), 4.21 (s, 2H), 2.92 (bs, 2H), 2.15 (bs,2H).

Example 68 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxyphenyl)pyrrolidine-2,5-dione

A mixture of3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxyphenyl)-1H-pyrrole-2,5-dione(0.73 g, 2.04 mmol), magnesium (0.89 g, 36.7 mmol) in anhydrous methanolwas heated to reflux for 1.5 h. After cooling to room temperature, thelight yellow solution was diluted with ethyl acetate (200 mL), washedwith 1.0 M hydrochloric acid (2×50 mL), water (100 mL), dried oversodium sulfate and concentrated to provide a light brown solid. Theresidue was purified by column chromatography on silica gel eluting with40-50% ethyl acetate in hexane to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxyphenyl)pyrrolidine-2,5-dioneas a light yellow solid. Mp 87-91° C.; ¹H NMR (CDCl₃) 400 MHz δ: 8.73(s. 1H), 7.25-7.30 (m, 1H), 7.14 (d, J=7.6 Hz, 1H), 6.93-7.01 (m, 3H),6.77-6.86 (m, 3H), 4.36 (d, J=6.4 Hz, 1H), 4.24 (d, J=6.4 Hz, 1H), 4.18(t J=5.5 Hz, 2H), 3.78 (s, 3H), 2.97 (t, J=5.6 Hz, 2H), 2.19-2.24 (m,2H).

Example 69 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-hydroxyphenyl)pyrrolidine-2,5-dione

To a solution of(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-methoxyphenyl)pyrrolidine-2,5-dionein dichloromethane (10 mL) at −78° C. under an atmosphere of nitrogenwas slowly added boron tribromide (1.0 M in dichloromethane) (5.2 mL).The resulting mixture was stirred at −78° C. for 30 minutes and at roomtemperature for 3 hours. The reaction mixture was cooled to −78° C. thenquenched by the addition of methanol (5 mL). The mixture was allowed towarm to room temperature and maintained at room temperature for 30minutes. The reaction mixture was diluted with dichloromethane (80 mL),washed with saturated aqueous sodium bicarbonate (15 mL), water (15 mL)and saturated sodium chloride (15 mL). The organic layer was dried overanhydrous sodium sulfate and concentrated to dryness. The residue waspurified by flash chromatography on silica gel eluting with ethylacetate:hexane:dichloromethane (5:5:1, v/v) to give(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(3-hydroxyphenyl)pyrrolidine-2,5-dioneas a brown solid (1.15 g, 63%); Mp 108-110° C. ¹H NMR (CDCl₃) 400 MHz δ:8.69 (s, 1H), 7.18 (t, 1H, J=8.0 Hz), 7.12 (d, 1H, J=8.0 Hz), 6.99 (d,1H, J=6.8 Hz), 6.97 (d, 1H, J=2.0 Hz), 6.93 (d, 1H, J=6.4 Hz), 6.76 (m,1H), 6.69 (d, 1H, J=1.6 Hz), 5.67 (brs, 1H), 4.32 (d, 1H, J=6.0 Hz),4.20 (d, 1H, J=6.0 Hz), 4.07 (t, 2H, J=5.6 Hz), 2.96 (t, 2H, J=6.0 Hz),2.19 (m, 2H), LC/MS: 347.3 [M+H].

Example 70 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-fluorophenyl)pyrrolidine-2,5-dione

3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-fluorophenyl)-1H-pyrrole-2,5-dioneprepared according to Example 67, was reduced by employing the method ofExample 68 to yield(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-fluorophenyl)pyrrolidine-2,5-dione1-ij]quinolin-1-yl)-4-(4-fluorophenyl)-1H-pyrrole-2,5-dione. Mp 208-210°C.; ¹H NMR (DMSO-d₆) 400 MHz δ: 11.52 (s. 1H), 7.40-7.43 (m, 2H), 7.32(m, 1H), 7.13-7.17 (m, 3H), 6.82-6.89 (m, 2H), 4.53 (m, 1H), 4.36 (m,1H), 4.09 (t, J=5.2 Hz, 2H), 2.89 (t, J=6.0 Hz, 2H), 2.09-2.11 (m, 2H).

Example 71 Preparation of(±)-Trans-3-[4-(benzyloxy)phenyl]-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)pyrrolidine-2,5-dione

3-[4-(benzyloxy)phenyl]-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-1H-pyrrole-2,5-dioneprepared according to Example 66, was reduced by employing the method ofExample 68 to yield (±)-trans3-[4-(benzyloxy)phenyl]-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)pyrrolidine-2,5-dione.Mp 91-93° C.; ¹H NMR (DMSO-d₆) 400 MHz δ: 11.47 (s. 1H), 7.25-7.43 (m,8H), 7.15 (d, J=7.6 Hz, 2H), 6.82-6.96 (m, 4H), 5.07 (s, 2H), 4.45 (d,J=7.6 Hz, 1H), 4.24 (d, J=7.6 Hz, 1H), 4.07-4.10 (m, 2H), 2.87-2.90 (m,2H), 2.09-2.10 (m, 2H).

Example 72 Preparation of(±)-Trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-hydroxyphenyl)pyrrolidine-2,5-dione

A mixture of(±)-trans-3-[4-(benzyloxy)phenyl]-4-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)pyrrolidine-2,5-dione(0.2 g) and Pd/C (10% w/w, 0.076 g) was stirred under 1 atmosphere ofhydrogen gas overnight. The catalyst was filtered off through a pad ofcelite and concentrated. The residue was purified by columnchromatography on silica gel eluting with 30-40% ethyl acetate in hexaneto provide(±)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(4-hydroxyphenyl)pyrrolidine-2,5-dione0.07 g as a light yellow solid. Mp 105-107° C.; ¹H NMR (acetone-d₆) 400MHz δ: 10.27 (s. 1H), 8.34 (s, 1H), 7.17-7.21 (m, 4H), 6.80-6.91 (m,4H), 4.39 (d, J=7.0 Hz, 1H), 4.22 (d, J=7.0 Hz, 1H), 4.13 (d, J=5.6 Hz,2H), 2.92 (d, J=5.2 Hz, 2H), 2.15-2.19 (m, 2H).

Example 73 Preparation of7-[4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester Step 1

To a solution of 7-formyl indole (2.4 g, 16.6 mmol) in1,2-dichloroethane (60 mL) was added aminoethanol (1.2 mL, 19.8 mmol)followed by glacial acetic acid (2.0 mL) and sodiumtriacetoxyborohydride (3.5 g, 16.6 mmol). The reaction mixture wasallowed to stir at room temperature for 16 hours. The reaction mixturewas quenched by addition of water (10 mL) and 1.0 M sodium hydroxide (10mL). The organic layer was then separated and the aqueous layerextracted with 1,2-dichloroethane (40 mL). The combined organic extractswere washed with saturated sodium bicarbonate (2×30 mL), water (2×50mL), dried over anhydrous sodium sulfate and evaporated to dryness.2-[(1H-indol-7-ylmethyl)-amino]-ethanol (4.4 g) was obtained as an oilLCMS (M+H)=189.

Step 2

To a solution of 2-[(1H-indol-7-ylmethyl)-amino]-ethanol (4.4 g) in1,2-dichloroethane (40 mL) was added triethylamine (4.85 mL, 34.6 mmol)followed by benzyl chloroformate (3.57 mL, 25.34 mmol). The mixture wasallowed to stir at room temperature for 2 hours. The mixture wasquenched by addition of water (20 mL), and 1.0 M sodium hydroxide (10mL). The organic layer was separated and the aqueous layer extractedwith 1,2-dichloroethane (20 mL). The combined organic extracts werewashed with 1.0 M hydrochloric acid (20 mL), water (20 mL), dried overanhydrous sodium sulfate and evaporated to dryness. The residue waspurified by silica gel chromatography, eluting with 20% ethyl acetate inhexanes to 40% ethyl acetate in hexanes to afford(2-hydroxy-ethyl)-(1H-indol-7-ylmethyl)-carbamic acid benzyl ester (2.79g, 52% combined yield for two steps) as a colorless oil. ¹H NMR (CDCl₃)400 MHz δ: 9.97 (br s, 1H), 7.75-6.9 (m, 8H), 6.54 (br s, 1H), 5.21 (s,2H), 4.9-4.6 (m, 3H), 3.85-3.57 (m, 2H), 3.55-3.23 (m, 3H); LCMSM+H=325.

Step 3

To a solution of (2-hydroxy-ethyl)-(1H-indol-7-ylmethyl)-carbamic acidbenzyl ester (2.79 g, 8.61 mmol) in dichloromethane (50 mL) was addedtriethylamine (1.56 mL, 11.2 mmol). The mixture was cooled to 0° C. andmethanesulfonyl chloride (0.74 mL, 9.47 mmol) added in a dropwisemanner. The mixture was warmed to room temperature and allowed to stirfor 2 hours. The mixture was then quenched with water (30 mL) and 1.0 Msodium hydroxide (10 mL). The organic layer was separated and theaqueous layer extracted with dichloromethane (20 mL). The combinedextracts were washed with 1.0 M hydrochloric acid (20 mL), water (30mL), dried over anhydrous sodium sulfate and evaporated to dryness.Methanesulfonic acid2-[benzyloxycarbonyl-(1H-indol-7-ylmethyl)-amino]-ethyl ester (3.46 g)was obtained as an oil Step 4

To a solution of methanesulfonic acid2-[benzyloxycarbonyl-(1H-indol-7-ylmethyl)-amino]-ethyl ester (3.46 g,8.61 mmol) in dimethylformamide (20 mL), which has been cooled to 0° C.was added sodium hydride (60%) in mineral oil. The reaction mixture wasallowed to stir at 0° C. for 1 hour and then quenched by the addition ofwater (40 mL). The aqueous layer was extracted with ethyl acetate (4×20mL). The combined organic extracts were washed with water (3×30 mL),dried over anhydrous sodium sulfate and evaporated to dryness. Theresidue was purified by silica gel chromatography, eluting withdichloromethane to afford3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylic acid benzylester (1.95 g, 74% combined yield for two steps) as a colorless oil. ¹HNMR (CDCL₃) 400 MHz δ: 7.6-7.45 (m, 1H), 7.4-7.2 (m, 5H), 7.17-6.9 (m,3H), 6.6-6.48 (m, 1H), 5.2-5.05 (m, 2H), 5.0-4.82 (m, 2H), 4.4-4.2 (m,2H), 4.1-3.95 (m, 2H); LCMS (M+H)=307.

Step 5

To a solution of3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylic acid benzylester (557 mg, 1.8 mmol), in anhydrous tetrahydrofuran (10 mL) at 0° C.,was added oxalyl chloride (238 W, 2.7 mmol) followed by a furtherportion of oxalyl chloride (340 W, 3.85 mmol). The mixture was stirredat 0° C. until all the starting material has been consumed before beingcooled to −78° C. Sodium methoxide in methanol (0.5M) (10 mL) was thenadded slowly and the mixture allowed to warm to room temperature. After1 hour at room temperature the mixture was then diluted with ethylacetate (200 mL) and washed with water (300 mL). The organic layer wasdried over anhydrous sodium sulfate and evaporated to dryness. Theresidue was purified by silica gel chromatography, eluting with a ethylacetate/hexanes (1:1) to afford7-methoxyoxalyl-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester as a pale yellow solid (481 mg, 67%). ¹H NMR (CDCl₃)400 MHz δ: 8.26-8.36 (m, 2H), 7.22-7.37 (m, 6H), 7.10 (dd, 1H, J=32.8and 7.2 Hz), 5.11 (d, 2H, J=8.0 Hz), 4.94 (d, 2H, J=22.4 Hz), 4.41-4.48(m, 2H), 4.01-4.05 (m, 2H), 3.93 (m, 3H).

Step 6

To a solution of7-methoxyoxalyl-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester (481 mg, 1.22 mmol) and indole-3-acetamide (234 mg,1.34 mmol) in anhydrous tetrahydrofuran (14 mL) at 0° C. was addedpotassium t-butoxide (412 mg, 3.67 mmol). The mixture was stirred at 0°C. for 2 hours. Concentrated hydrochloric acid (5 mL) was then added andthe mixture stirred for 2 hours at room temperature. The mixture wasthen diluted with ethyl acetate (300 mL), washed with water (500 mL),and the organic layer dried over anhydrous sodium sulfate. The residuewas purified by silica gel chromatography, eluting with a ethylacetate/hexanes (1:1) to afford7-[4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester as a bright orange/red solid (1.2 g, 80%). ¹H NMR(DMSO-d₆) 400 MHz δ: 11.66 (d, 1H, J=2.4 Hz), 10.94 (s, 1H), 7.69-7.75(m, 2H), 7.19-7.38 (m, 6H), 6.98 (t, 1H, J=7.2 Hz), 6.73-6.89 (m, 3H),6.60-6.66 (m, 2H), 4.90-5.08 (m, 2H), 4.50 (m, 2H), 3.95 (m, 2H).

Example 74 Preparation of(±)-Trans-7-[4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-3-yl]-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester

Magnesium turnings (195 mg, 8.0 mmol) were added to a solution of7-[4-(1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester (230 mg, 0.44 mmol) in anhydrous methanol (20 mL) andheated to reflux under an atmosphere of nitrogen for 1.5 hours. Aftercooling to room temperature the mixture was poured into ethyl acetate(200 mL) and washed with 1 M hydrochloric acid (100 mL). The organiclayer was dried over anhydrous sodium sulfate and evaporated to dryness.The residue was then purified by silica gel chromatography using 50-60%ethyl acetate in hexanes to yield(±)-trans-7-[4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-3-yl]-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester as an off white solid (205 mg). ¹H NMR (DMSO-d₆) 400MHz δ: 11.56 (s, 1H), 11.03 (d, 1H, J=2 Hz), 7.21-7.43 (m, 10H), 7.09(t, 1H, J=7.2 Hz), 6.92-7.00 (m, 2H), 6.82-6.89 (m, 3H), 5.04 (s, 2H),4.87 (d, 2H, J=7.6 Hz), 4.54 (dd, 2H, J=7.6 and 28.8 Hz), 4.30 (m, 2H),3.92 (m, 2H).

Example 75 Preparation of(±)-Trans-3-(1H-indol-3-yl)-4-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-pyrrolidine-2,5-dione

(±)-Trans-7-[4-(1H-indol-3-yl)-2,5-dioxo-pyrrolidin-3-yl]-3,4-dihydro-1H-[1,4]diazepino[6,7,1-hi]indole-2-carboxylicacid benzyl ester (161 mg, 0.31 mmol) and 10% palladium on carbon (100mg) in anhydrous methanol (15 mL) were stirred under 1 atmosphere ofhydrogen for 16 hours. The catalyst was then filtered through a bed ofCelite and the filtrate evaporated to dryness to yield(±)-trans-3-(1H-indol-3-yl)-4-(1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]indol-7-yl)-pyrrolidine-2,5-dioneas an off white solid (95 mg). ¹H NMR (DMSO-d₆) 400 MHz δ: 11.04 (d, 1H,J=1.6 Hz), 7.35-7.42 (m, 4H), 7.24 (dd, 1H, J=2.8 and 5.6 Hz), 7.09 (t,1H, J=7.2 Hz), 6.89-6.98 (m, 3H), 4.52 (dd, 2H, J=7.2 and 24.8 Hz), 4.11(s, 2H), 4.07-4.10 (m, 2H), 3.14-3.17 (m, 2H).

Example 76 Combination of c-Met Inhibitors with Sorafenib and Sunitinibfor the Treatment of Various Anti-Proliferative Disorders and Cancer

Materials and Methods

Unless otherwise stated, the following materials and methods apply tothe biological assays described herein.

Cell culture and reagents: NCI-H441, PC-3, MIA PaCa-2, HeLa, HT-29 andA549 cancer cell lines (American Type Culture Collection) were culturedin Dulbecco's modified Eagle's medium containing 10% fetal bovine serum,100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine.

Isobologram analysis: For each cell line and drug combination, the 72hour IC₅₀ values were determined for each individual drug and incombination at the equipotent fixed ratio by MTT proliferation endpointassay or by colony formation assay. For example, in the case of drugs Aand B, where the IC₅₀ values are 1 μM and 5 μM, respectively, theequipotent ratio is 1:5. Therefore, a serial dilution of the highestcombination concentration (8× to 0.125X, where X is the IC₅₀ ratioconcentration) was used to generate a dose response curve. The degree ofinhibition of cell proliferation in this assay relative to unexposedcontrols was designated the “effect”, which ranged from 0.0 (noinhibition) to 1.0 (no cellular conversion of the MTT or MTS reagent,denoting complete cell death). Duplicate independent experiments wereperformed for each cell line/drug combination. Data were then analyzedby Calcusyn™ (Biosoft, Cambridge, UK) data analysis software.

Cell survival analysis. In some experiments, cell survival wasdetermined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) assay. Briefly, cells were plated in a 96-well plate at5-10,000 cells per well, cultured for 24 hours in complete growthmedium, and then treated with various drugs and drug combinations for 72hours. MTT was added to a final concentration of 0.5 mg/mL, andincubated for 1 hour, followed by assessment of cell viability using themicroplate reader at 570 nm. Data were normalized to untreated controlsand analyzed with Microsoft Excel.

Cell proliferation assay. Exponentially growing cells were seeded at2,000 cells per well in 6-well plates and allowed to attach for 24hours. Increasing concentrations of individual drugs and those incombination were then added to the media for another 24 hours. After 24hours exposure, the drug was removed and fresh media was added for thenext 14-21 days, allowing for colony formation. Cells were fixed andstained with GIEMSA (Gibco BRL). Colonies of greater than 50 cells werescored as survivors and the percentage of cell survival was plotted todetermine the IC₅₀ values.

The studies described herein used a compound of Formula Va shown herein,namely,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione,a small molecule inhibitor of the c-Met receptor tyrosine kinase, incombination with the multi-targeted kinase inhibitor, sorafenib.

A panel of 64 human cancer cell lines encompassing a spectrum ofgenotypes and tissue origins were surveyed in 72 h MTS cytotoxicityassays across a wide range of compound concentrations.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione and sorafenib were configured in checkerboard3-fold dilutions for a 72-hr MTS assay.

In the instant example, two independent experiments were performed inparallel. The Chou algorithm was employed to calculate the CombinationIndex (CI) which is shown in Table 1.

TABLE 1 Criteria for Combination Index (CI) CI ≦ 0.3 Strong Synergy 0.3< CI ≦ 0.85 Synergy 0.85 < CI ≦ 1.2 Additive 1.2 < CI ≦ 3.3 AntagonismCI ≧ 3.3 Strong Antagonism

Combination indices were determined according to the method of Chou.{Chou, T.-C. 1991. The median-effect principle and the combination indexfor quantitation of synergism and antagonism, p. 61-102. In T.-C. Chouand D. C. Rideout (ed.), Synergism and antagonism in chemotherapy.Academic Press, San Diego, Calif}. FIG. 2, panel A, shows where C_(A)^(O) and C_(B) ^(O) are the separate concentrations of drug A and Brespectively that achieve the same effect as the mixture of drug A andof drug B. Isobologram analyses categorized drug combinations assynergistic, additive, or antagonistic. Specifically, FIG. 2, panel B,shows isobologram analyses for a particular effect (e.g., 50% of themaximum) in which the dose of drug A alone is A=20 and drug B alone isB=100. The straight line connecting these intercept points is theadditivity line which, based on these potencies, should give the sameeffect. An actual dose pair such as point Q attains this effect withlesser quantities and is supra-additive or synergistic, while the dosepair denoted by point R means greater quantities are required and istherefore sub-additive. A point such as P that appears below the linewould be simply additive.

The combination data of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewith sorafenib are shown in Table 2 and the combination data withsunitinib are shown in Table 3. The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” in these tables. The identity and tissueorigin of cancer cell lines are indicated. The results show that thecombination(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib revealed synergistic cytotoxicity in 3 NSCLC cell lines(NCI-H522, NCI-H358, NCI-H460), MDA-MB-231 (breast), A375 (melanoma),the HCC1395 breast cancer line, the Caki-1 renal cell carcinoma, theHeLa cervical carcinoma cell line, and the A431 epidermoid carcinoma andshowed additive cytotoxicity in 40 other cell lines including, but notlimited to, the Colo205 and SW480 colon cancer lines, the NCI-H358(NSCLC) cell line, and 3 hepatocellular carcinomas (JHH-4, PLC/PRF/5,SK-Hep-1). Table 2 shows the combination cytoxicity of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib in human cancer cell lines in which either additivity orsynergism (supra-additivity) were observed. Table 3 shows thecombination cytoxicity of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sunitinib in human cancer cell lines in which either additivity orsynergism (supra-additivity) were observed.

TABLE 2 Human Cancer Cell Tissue of Combination Effect Line Origin(Combination Index) Agent A and sorafenib MDA-MB-231 Breast Synergistic(0.61) A375 Melanoma Synergistic (0.63) NCI-H522 Lung Synergistic (0.65)(NSCLC) HeLa Cervix Synergistic (0.66) Caki-1 Renal Synergistic (0.71)NCI-H358 Lung Synergistic (0.74) (NSCLC) NCI-H460 Lung Synergistic(0.80) (NSCLC) A431 Skin Synergistic (0.83) HCC1395 Breast Synergistic(0.85) NCI-H1299 Lung Additive (0.87) (NSCLC) COLO205 Colon Additive(0.88) HeLa S3 Cervix Additive (0.92) SNU-16 Stomach Additive (0.93)SW480 Colon Additive (0.94) PLC/PRF/5 Liver Additive (0.94) (hepatoma)NCI-H1993 Lung Additive (0.96) (NSCLC) KATO III Stomach Additive (0.96)SCH Stomach Additive (0.98) A549 Lung Additive (0.98) (NSCLC) MKN45Stomach Additive (1.00) K562 Blood Additive (1.01) HCT116 Colon Additive(1.02) SK-OV-3 Ovary Additive (1.02) DMS 53 Lung Additive (1.05) 786-ORenal Additive (1.05) WI-26 VA4 Lung Additive (1.05) MDA-MB-453 BreastAdditive (1.06) T98G Brain Additive (1.06) MDA-MB-361 Breast Additive(1.07) SK-Hep-1 Liver Additive (1.07) (hepatoma) DU145 Prostate Additive(1.08) NCI-H1581 Lung Additive (1.09) (NSCLC) LN-18 Brain Additive(1.10) DLD-1 Colon Additive (1.10) WM-266-4 Skin Additive (1.11) C-33ACervix Additive (1.12) LoVo Intestine Additive (1.12) JHH-4 LiverAdditive (1.13) (hepatoma) AGS Stomach Additive (1.13) G-361 SkinAdditive (1.13) ZR-75-1 Breast Additive (1.14) MDA-MB-468 BreastAdditive (1.16) Mia PaCa-2 Pancreas Additive (1.17) HLF Liver Additive(1.17) (hepatoma) U937 Blood Additive (1.18) SNU-398 Liver Additive(1.18) (hepatoma) SW620 Colon Additive (1.19) NCI-H69 Lung Additive(1.19) SK-BR-3 Breast Additive (1.19)

TABLE 3 Human Cancer Cell Tissue of Combination Effect Line Origin(Combination Index) Agent A & sunitinib SCH Stomach Synergistic (0.78)SNU-16 Stomach Synergistic (0.84) KATO III Stomach Additive (1.02)NCI-H1581 Lung (NSCLC) Additive (1.04) DLD-1 Colon Additive (1.07) LoVoIntestine Additive (1.08) G-402 Kidney Additive (1.09) K562 bloodAdditive (1.12) MDA-MB- Breast Additive (1.12) 453 DMS 53 Lung Additive(1.15) LN-18 Brain Additive (1.15) U937 blood Additive (1.15) HCT116Colon Additive (1.16) NCI H1703 Lung (NSCLC) Additive (1.19) HeLa CervixAdditive (1.20) SNU-5 Stomach Additive (1.20)

The anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib on the NCI-H522 NSCLC cell line in vitro was alsoassessed. Cells were treated with increasing concentrations of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionefrom 0.14 to 33 μM and sorafenib from 0.015 to 100 μM for 72 hr. Cellgrowth was assessed using a standard commercially available MTS reagent[3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt fromPromega, Madison, Wis.] assay. An isobologram was plotted and thecombination index was determined which showed the synergism of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib.

The in vivo anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib was also assessed. Female Ncr nu/nu mice with establishedsubcutaneous NCI-H522 NSCLC tumors were treated by oral gavage with theindicated doses of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,sorafenib, both agents, or vehicle control for 21 days (days 8-29). Allregimens were orally administered once daily for 21 days. Tumor sizeswere evaluated periodically during treatment at the indicated dayspost-inoculation. FIG. 3 is a graphical representation of thecombination treatment of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(shown as Agent A) and sorafenib demonstrating an effect in the NCI-H522NSCLC xenograft model. These results are represented as the mean oftumor weight±SEM of 10 tumors during the treatment period. Bothcompounds were well-tolerated in all cohorts, and no adverse effects onbody weight gain were observed, showing that these two agents areeminently combinable in vivo.

Taken together, these pre-clinical data demonstrate that thecombinational therapy of c-Met inhibitors, such as(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,and kinase inhibitors, such as sorafenib, shows highly encouraginganti-proliferative activity against a wide range of cancer cell lines,in vitro and in vivo.

Example 77 Combination of c-Met Inhibitors and Sorafenib for theTreatment of Microphthalmia Transcription Factor-associated Tumors

In clinical studies, monotherapy treatment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionehas been well tolerated and has resulted in tumor responses andprolonged stable disease across broad ranges of tumors and doses.Indications with favorable clinical treatment include, MiT(Microphthalmia Transcription Factor)-associated tumors, non-small celllung cancer and pancreatic adenocarcinoma. MiT tumors, which includeclear cell sarcoma (CCS), alveolar soft part sarcoma (ASPS) andtranslocation-associated renal cell carcinoma (RCC), are linkedbiologically through a common chromosomal abnormality that isresponsible for the over-expression of c-Met resulting in thedevelopment of these tumors. Similar clinical studies are directed tohepatocellular carcinoma (HCC) both for monotherapy treatment andcombinational therapy with kinases inhibitors, such as sorafenib.

According to the National Cancer Institute, 21,370 new cases of HCC inthe United States were projected in 2008, and 18,410 deaths wereprojected to be caused by the disease. In the U.S., the increasingincidence of HCC is related primarily to hepatitis C infection andcirrhosis. The first drug to be approved for patients with unresectableHCC was sorafenib. For patients who experience disease progressionfollowing sorafenib treatment or for those patients unable to toleratesorafenib, no alternative therapy with proven clinical benefit isavailable. Thus, there is a high unmet medical need for novel treatmentapproaches in patients with advanced HCC for whom sorafenib treatment isnot an option.

Over-expression of c-Met and its ligand, hepatocyte growth factor (HGF),is associated with poor prognoses in patients with HCC. When abnormallyactivated, c-Met plays multiple roles in aspects of human cancer,including cancer cell growth, survival, angiogenesis, invasion andmetastasis. Scientific literature related to HCC provides evidence ofthe aberrant activation of the c-Met pathway. In addition, thedysregulation of c-Met and HGF has been shown to be common in thisdisease. Cell proliferation is a central mechanism responsible for livercancer progression, and c-Met is believed to play an important role inthis process.

In these clinical studies combining(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib, Patients are treated with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneat 360 mg twice daily (bid) and sorafenib at 200 mg bid. Patients arealso screened to valuate dynamic changes of hepatocyte growth factor(HGF), vascular endothelial growth factor (VEGF), and soluble c-Met inpatients' peripheral blood that are associated with treatment of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib. The results of the combinational treatment show additiveand synergistic anti-proliferative effects when compared to monotherapytreatment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

Example 78 Combination of c-Met Inhibitors and Kinase Inhibitors for theTreatment of Various Anti-Proliferative Disorders and Cancer

To complement Phase I data showing clinical response as a single agent,the potential synergy of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dionewith two marketed tyrosine kinsase inhibitors (TKIs), sorafenib (alsoshown in Examples 76 and 77) and sunitinib was assessed. A panel of 64human cancer cell lines encompassing a spectrum of genotypes and tissueorigins were surveyed in 72 hour MTS cytotoxicity assays across a widerange of compound concentrations. Isobologram analyses categorized drugcombinations as synergistic, additive, or antagonistic. The combinationof(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib revealed synergistic cytotoxicity in 3 NSCLC cell lines(NCI-H522, NCI-H358, NCI-H460), MDA-MB-231 (breast), A375 (melanoma),the HCC 1395 breast cancer line, the Caki-1 renal cell carcinoma, theHeLa cervical carcinoma cell line, and the A431 epidermoid carcinoma(see Example 89). Additivity was seen in 40 human cancer cell lines withthe(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione/sorafenibcombination including, but not limited to the Colo205 and SW480 coloncancer lines, the NCI-H358 (NSCLC) cell line, and 3 hepatocellularcarcinomas (JHH-4, PLC/PRF/5, SK-Hep-1). This data may be informative inthe design of combination therapy regimens for(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.

In vitro combination cytotoxicity studies were conducted with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,in combination with sorafenib and sunitinib against a large panel ofhuman cancer cell lines. A wide range of combination effects wasobserved, with a higher incidence of either synergistic or additivecytotoxic effects documented with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib as compared with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sunitinib. Interestingly, the two cell lines where(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sunitinib showed synergistic cytotoxicity are both known to expressactivated c-Met due to c-Met gene amplification (the SNU-16 gastriccarcinoma and the SCH gastric choriocarcinoma cell line) (Table 3). Thecompound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table. While the effects observedwere not tissue-type specific, the cell lines exhibiting synergy with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib included two c-Met expressing breast carcinoma cell lines(HCC1395 and MDA-MB-231) and three non-small cell lung carcinomas(NSCLC), NCI-H460, NCI-H358 and NCI-H522, all with documented c-Metexpression or gene amplification. The NCI-H522 NSCLC cell line is knownto exhibit c-Met gene amplification and secrete high levels of HGF, butit does not exhibit high constitutive levels of phospho-c-Met upon serumstarvation. Nonetheless,(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib demonstrated augmented anti-tumor efficacy whenco-administered orally in an athymic mouse xenograft model (FIG. 3).(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand sorafenib showed additive cytotoxicity in 5 hepatocellularcarcinomas (HCC) tested, a clinical indication for which sorafenib iscurrently approved. Recapitulation of the synergistic or additivecytotoxic effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneand marketed TKIs in relevant animal models guide potential clinicaldevelopment strategies for(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,a novel, orally administered and well-tolerated anti-cancer drugcandidate that demonstrates indications of clinical activity.

The combination data of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione(shown as Agent A in FIG. 4) with either sorafenib or sunitinib wereexpressed as 1/Combination Index as shown in FIGS. 4 and 5.

Example 79 Combination of c-Met Inhibitors and Erlotinib for theTreatment of Non-Small Cell Lung and Colon Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith erlotinib were tested in NCI-H441 non-small cell lung cancer cells(NSCLC). Erlotinib is an inhibitor of the epidermal growth factorreceptor and is indicated for the treatment of patients with locallyadvanced or metastatic non-small cell lung cancer after failure of atleast one prior chemotherapy regimen, and is indicated for thefirst-line treatment of patients with locally advanced, unresectable ormetastatic pancreatic cancer. The IC₅₀ of erlotinib was predicted to beapproximately 1 μM, while the IC₅₀ of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas predicted to be 300 nM for NCI-H441 NSCLC cells and therefore an(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:erlotinibratio of 1:3 was used. A 1:33(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:erlotinibwas used for HT29 colon cancer cells. The CI ranged between 0.45-1 atthe ED₅₀ for the NCI-H441 cell line as determined by independentexperiments, and the CI was 0.72 for the Ht29 cell line (Table 4). Thecompound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” in this table. Median effect plots andisobolograms were performed for each experiment. This data demonstratesan additive to synergistic anti-proliferative effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand erlotinib in both non-small cell lung cancer cells (NSCLC) and coloncancer cells.

TABLE 4 Combination Index values at Cell Lines and Agent(s) ED25 ED50ED75 IC₅₀ r NCI-H441 Agent A & Erlotinib (1:3) 0.96 0.45 0.30  277 nM0.98 Agent A N/A N/A N/A 1477 nM 0.94 Erlotinib N/A N/A N/A 3159 nM 0.99NCI-H441 Agent A & Erlotinib (1:3) 2.02 1.01 0.52  881 nM 0.98 Agent AN/A N/A N/A 3305 nM 0.97 Erlotinib N/A N/A N/A 3562 nM 0.98 HT29 Agent A& Erlotinib (1:33) 1.03 0.72 0.51  117 nM 0.96 Agent A N/A N/A N/A  179nM 0.97 Erlotinib N/A N/A N/A 56334 nM  0.98

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith erlotinib were tested in a NCI-H441 NSCLC human tumor xenograftmodel.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas administered daily at 300 mg/kg orally, five days a week for fourweeks (qd×5×4). Erlotinib was administered daily at 100 mg/kg or 50mg/kg orally, five days a week for four weeks. As shown in Table 5 andFIG. 6, in all studies, the combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith erlotinib showed improved anti-proliferative effects overmonotherapy treatment and the data demonstrates at least an additive,and possibly synergistic, effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith erlotinib in non-small cell lung cancer. The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” Table 5 and FIG. 6.

TABLE 5 Dosage T/C Min BW loss (mg/kg) Route Schedule (on day) (g)Mortality Non-Treatment — — — 1.00 — 0/5 Agent A 300 p.o. qdx5x4 0.52(17) — 0/5 Erlotinib 100 p.o. qdx5x4 0.50 (17) −1.0 (4) 0/5 50 p.o.qdx5x4 0.59 (17) −0.6 (4) 0/5 Agent A + 300 p.o. Day 0-4, Day 7*Erlotinib 100 p.o. Day 0-4, Day 7* 0.43 (10) −3.5 (7) 0/5 Agent A + 300p.o. qdx5x4 Erlotinib 50 p.o. qdx5x4 0.27 (17)  −1.2 (10) 0/5 *Drugadministration after day 8 was terminated due to >10% body weight loss.

Example 80 Combination of c-Met Inhibitors and Gefitnib for theTreatment of Colon Cancer and Lung Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gefitinib were tested in HT29 colon cancer cells. Gefitinib is aninhibitor of the EGF receptor. The IC₅₀ of gefitinib was predicted to beapproximately 5 μM, while the IC₅₀ of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas predicted to be 150 nM for HT29 colon carcinoma cells, therefore an(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:gefitinibratio of 1:33 was used. The CI was 1.27 at the ED₅₀ (Table 6). Thecompound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table. Median effect plots andisobolograms were performed for each experiment. This data demonstratesat least an additive anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gefitinib in colon cancer cells.

TABLE 6 Combination Parameters CI at Agent(s) ED₅₀ IC₅₀ r Agent A &Gefitinib (1:33) 1.27  99 nM 0.97 Agent A N/A 116 nM 0.93 Gefitinib N/A7803 nM  0.97

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gefitinib were tested in a NCI-H441 human lung tumor xenograftmodel.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas administered daily at 300 mg/kg orally, five days a week for fourweeks (qd×5×4). Gefitinib was administered daily at 50 mg/kg orally,five days a week for four weeks. As shown in Table 7 and FIG. 7, in allstudies, the combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gefitinib showed improved anti-proliferative effects overmonotherapy treatment and the data demonstrates at least an additive,and possibly synergistic, effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gefitinib in non-small cell lung cancer. The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” Table 7 and FIG. 7.

TABLE 7 BW Dosage T/C Min loss (mg/kg) Route Schedule (on day) (g)Mortality Non- — — — 1.00 — 0/5 Treatment Agent A 300 p.o. qdx5x4 0.52(17) — 0/5 Gefitinib 50 p.o. qdx5x4 0.60 (21) −0.5 (4) 0/5 Agent A + 300p.o. qdx5x4 Gefitinib 50 p.o. qdx5x4 0.40 (21) −1.0 (4) 0/5

Example 81 Combination of c-Met Inhibitors and Carboplatin for theTreatment of Pancreatic Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith the DNA synthesis inhibitor carboplatin, was tested in MIA PaCa-2pancreatic tumor cells. Carboplatin has been reported to be beneficialin both pancreatic and prostate cancer when combined with othertherapeutic agents. The IC₅₀ of carboplatin was predicted to beapproximately 25-50 μM, while the IC₅₀ of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas predicted to be 150 nM for MIA PaCa-2 cells. An(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:carboplatinratio of 1:50 was used, unless otherwise indicated, due to theinsolubility of carboplatin at higher concentrations. For MIA PaCa-2cells the CI ranged between 1.09-1.45 at the 50% effective dose (ED₅₀)(Table 8). The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table. Median effect plots andisobolograms were performed for each experiment. This data demonstratesat least an additive anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith carboplatin in pancreatic cancer cells. The mild variabilityobserved in this drug combination is likely due to solubility issues ofcarboplatin at the higher dose ranges.

TABLE 8 Combination Parameters Agent(s) CI at ED₅₀ IC₅₀ R Agent A &Carboplatin 1.09 0.11 μM 0.99 (1:80) Agent A N/A   49 μM 0.96Carboplatin N/A 0.12 μM 0.97 Agent A & Carboplatin 1.16 0.12 μM 0.96(1:50) Agent A N/A 0.13 μM 0.96 Carboplatin N/A   28 μM 0.97 Agent A &Carboplatin 1.45 0.18 μM 0.98 (1:50) Agent A N/A 0.15 μM 0.95Carboplatin N/A   37 μM 0.98

Example 82 Combination of c-Met Inhibitors and Cisplatin for theTreatment of Pancreatic Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith the DNA synthesis inhibitor cisplatin were tested in MIA PaCa-2pancreatic tumor tumor cells. The IC₅₀ of cisplatin was predicted to beapproximately 5-15 μM, while the IC₅₀ of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas predicted to be 150 nM for MIA PaCa-2 cells. An(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:cisplatinratio of 1:50 was used unless otherwise indicated. For MIA PaCa-2 cellsthe CI ranged between 0.74-0.79 at the ED₅₀ (Table 9). The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table. Median effect plots andisobolograms were performed for each experiment. This data demonstratesa synergistic anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand cisplatin in pancreatic cancer cells.

TABLE 9 Combination Parameters Agent(s) CI at ED₅₀ IC₅₀ r Agent A &Cisplatin 0.79 0.040 μM  0.95 (1:50) Agent A N/A 0.17 μM 0.93 CisplatinN/A 3.79 μM 0.97 Agent A & Cisplatin 0.74 0.05 μM 0.95 (1:42) Agent AN/A 0.29 μM 0.95 Cisplatin N/A 3.37 μM 0.96

Example 83 Combination of c-Met Inhibitors and Various ChemotheraputicAgents for the Treatment of Gatric Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith 5-FU, TS-1, Capecitabine, and cisplatin (CDDP) were tested inMKN-45 human gastric tumor xenograft model (FIG. 11-14).(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas administered daily at 300 mg/kg orally, five days a week for twoweeks (qd×5×2). 5-FU was administered daily at 10 mg/kg intravenously,five days a week for two weeks. TS-1 was administered daily at 10 mg/kgorally, five days a week for two weeks. Capecitabine was administereddaily at 360 mg/kg orally, five days a week for two weeks. CDDP wasadministered weekly at 5 mg/kg intravenously for two weeks. As shown inTable 10, in all studies, the combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith these compounds showed improved anti-proliferative effects overmonotherapy treatment and the data demonstrates at least and additiveanti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith 5-FU, TS-1, Capecitabine, and cisplatin in gastric cancer.

TABLE 10 Dosage T/C Min BW loss Mor- (mg/kg) Route Schedule (on day) (g)tality Non- — — — 1.00 −2.3 (21) — Treatment Agent A 300 p.o. qdx5x20.50 (4) −1.7 (21) 0/5 5-FU 10 i.v. qdx5x2 0.63 (14) −3.4 (21) 0/5 TS-110 p.o. qdx5x2 0.45 (10) −3.6 (14) 1/5 Capecitabine 360 p.o. qdx5x2 0.42(14) −1.4 (21) 0/5 Cisplatin 5 i.v. Weeklyx2 0.55 (10) −2.0 (14) 0/5(CDDP) Agent A + 300 p.o. qdx5x2 5-FU 10 i.v. qdx5x2 0.31 (14) −0.9 (10)0/5 Agent A + 300 p.o. qdx5x2 TS-1 10 p.o qdx5x2 0.29 (14) −1.3 (21) 0/5Agent A + 300 p.o. qdx5x2 Capecitabine 360 p.o. qdx5x2 0.29 (14) −1.2(4)  0/5 Agent A + 300 p.o. qdx5x2 Cisplatin 5 i.v. weeklyx2 0.43 (14)−2.0 (10) 0/5

Example 84 Combination of c-Met Inhibitors and Imatinib for theTreatment of Colon Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith imatinib (Gleevec) were tested in HT29 colon cancer cells. Imatinibis an inhibitor of the Abelson proto-oncogene, c-kit, and PDGF-R(platelet-derived growth factor receptor) and is indicated for thetreatment of chronic myelogenous leukemia (CML), gastrointestinalstromal tumors (GISTs) and a number of other malignancies. The IC₅₀ ofimatinib was predicted to be approximately 10 μM, while the IC₅₀ of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas predicted to be 150 nM for HT29 cells, therefore an(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:imatinibratio of 1:66 was used. The CI was 1.22 at the ED₅₀ (Table 11). Thecompound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table. Median effect plots andisobolograms were performed for each experiment. This data demonstratesa nearly additive anti-proliferative effect (a CI of 1.22 nearly meetingthe criterion of a CI=1.2) of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand imatinib in colon cancer cells.

TABLE 11 Combination Parameters Agent(s) CI at ED₅₀ IC₅₀ r Agent A &Imatinib 1.22  70 nM 0.98 (1:66) Agent A N/A 118 nM 0.96 Imatinib N/A7374 nM  0.99

Example 85 Combination of c-Met Inhibitors and Gemcitabine for theTreatment of Pancreatic Cancer

c-Met was first discovered in the 1980s as an activated oncogene(Cooper, C. S. et al. (1984). Nature 311, 29-33) and is the prototypemember of a sub-family of RTKs, including Ron, that are structurallydistinct from other RTK families. c-Met is the only known high-affinityreceptor for hepatocyte growth factor (HGF), also known as scatterfactor (Birchmeier, C. et al. (2003). Nat Rev Mol Cell Biol 4, 915-925).In vitro and in vivo experiments have shown that this receptor-growthfactor pair is involved in multiple physiologic cellular responsesincluding embryogenesis, cell proliferation, survival, differentiation,motility, and invasion (Birchmeier, C. et al. (2003). Nat Rev Mol CellBiol 4, 915-925). Subsequently, HGF and/or c-Met have been found to befrequently over-expressed in many types of human solid tumors, includingsarcomas and carcinomas, and in their associated metastases where thedegree of c-Met expression often correlates with poor patient prognosis(Birchmeier, C. et al. (2003). Nat Rev Mol Cell Biol 4, 915-925).Activating c-Met mutations have been described in both sporadic andinherited forms of human renal papillary carcinomas(Danilkovitch-Miagkova, A., and Zbar, B. (2002). J Clin Invest 109,863-867), while genomic amplification of met has been found associatedwith gastric carcinoma (Nakajima, M. et al. Cancer 85, 1894-1902).Ectopic HGF and/or c-Met overexpression can drive tumorigenesis andmetastasis in both human xenograft tumor bearing mice and transgenicmouse models (Takayama, H. et al. (1997). Proc Natl Acad Sci USA 94,701-706). Taken together, these data provide compelling evidence for thefunctional relevance of c-Met activated networks in tumorigenesis andmetastatic progression, thus c-Met is an attractive cancer therapeutictarget.

(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionehas been shown to inhibit c-Met activity in biochemical assays and in anumber of cell-based assays.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas advanced into clinical trials and was well-tolerated, exhibitingsigns of tumor response in late stage cancer patients with metastaticdisease (Rosen, L. et al. (2006) 18^(th) EORTC-NCI-AACR Symposium onMolecular Targets and Cancer Therapeutics (7-10 Nov. 2006, Prague, CzechRepublic). Eur J Cancer Suppl 4, 196).

To potentially inform phase II clinical trials, in vitro combinationstudies were performed using(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gemcitabine, the current drug standard of care used for pancreaticcancer treatment. The purpose of this study was to independentlyrecapitulate the effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gemcitabine combination on human pancreatic cancer cell lines usinga different method of calculation and comparing with the data obtainedby the CalcuSyn™ software (Biosoft).

MIA PaCa-2 (also referred to as PACA2), PANC-1, CFPAC-1, and Hs766Thuman pancreatic cell lines were maintained in DMEM supplemented with10% fetal bovine serum (FBS), penicillin, streptomycin, and fungizone.AsPC-1 cells were maintained in RPMI supplemented with 10% FBS,penicillin, streptomycin, and fungizone. HPAF-II cells were maintainedin MEM supplemented with 10% FBS, penicillin, streptomycin, andfungizone. For the MTS cytotoxicity assay, cells were plated in 96-wellplates at 2,000 cells per well and incubated with increasingconcentrations of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gemcitabine in combination for 72 hr. MTS reagent (Promega, Madison,Wis.) was added to each well and plates were incubated for 4 hr at 37°C. The absorbance of each well was measured at 492 nm using a microplatereader. A preliminary experiment with(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneor gemcitabine (GEM) alone was performed to determine the IC₅₀ of eachindividual compound on each cell line and a concentration range was alsodetermined, The layout of dose range of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione(shown as Agent A in FIG. 8) and gemcitabine for drug combinationanalysis was further determined. The IC₅₀ calculation and IC₅₀ valuedeterminations are shown in Table 12 and FIG. 8.

The results of these studies show that the combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gemcitabine provides a synergistic anti-proliferative effect inPANC-1, HPAF-II and AsPC-1 human pancreatic cancer cell lines. All threepancreatic cell lines were sensitive to the combinatorial treatment of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gemcitabine. The results of this study recapitulate previous workand confirm that the combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gemcitabine is synergistic in 3 out of the 6 pancreatic cell linestested.

TABLE 12 Combination Tissue Chemotherapeutic Index Values CombinationCell Line Origin Agent at ED₅₀ Conclusion AsPC-1 Pancreatic Gemcitabine0.6 Synergism HPAF-II Pancreatic Gemcitabine 0.2 Synergism PANC-1Pancreatic Gemcitabine 0.7 Synergism

Example 86 Combination of c-Met Inhibitors and Taxotere for theTreatment of Pancreatic Cancer, Colon Cancer and Prostate Cancer

The effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith taxotere were tested in MIA PaCa-2 pancreatic tumor cells, PC-3prostate tumor. For each cell line, the IC₅₀ value of taxotere waspredicted to be approximately 5 nM, while the IC₅₀ of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas predicted to be 150 nM for MIA PaCa-2 cells, and 1 μM for PC-3cells. For these cell lines(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:taxotereratios of 200-1000:1 were used, depending on predicted IC₅₀ values. ForMIA PaCa-2 cells the CI was 0.99 at the ED₅₀, (Table 15) and thereforethe effects of the combination were additive. The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table. Median effect plots andisobolograms were performed for each experiment.

TABLE 15 Combination Parameters Agent(s) CI at ED₅₀ IC₅₀ R Agent A &Taxotere (200:1) 0.99 149 nM 0.94 Agent A N/A 177 nM 0.95 Taxotere N/A 5.1 nM 0.99

This combination was also tested in PC-3 cells, where the combinationindex ranged between 0.68 and 1.51 at ED₅₀ (Table 16). The compound(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneis identified as “Agent A” is this table.

TABLE 16 Combination Parameters Agent(s) CI at ED₅₀ IC₅₀ R Agent A &Taxotere (200:1) 1.51 590 nM 0.98 Agent A N/A 875 nM 0.98 Taxotere N/A 3.5 nM 0.97 Agent A & Taxotere (200:1) 0.68 259 nM 0.93 Agent A N/A 553nM 0.96 Taxotere N/A  6 nM 0.96

It has been demonstrated that(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand taxotere has a beneficial effect when dosed in combination inxenograft studies. While the 72 hour MTT data suggested that thecombination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand taxotere was additive, the MTT assay may be the most accurate assayof cell death due to taxotere's mechanism of action of cell death.Therefore, additional combination cell death assays using colonyformation were performed in order to capture the long term effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand taxotere on cell death. Colony formation assays were performed inPC-3, HT29, and MIA PaCa-2 cells to determine whether the effects ofthis combination were synergistic. By colony formation assay the CI forMIA PaCa-2 cells was 0.43, which indicates synergism. Slight synergismto additivity was also observed for HT-29 cells, whereas the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione:taxoteremixture was additive for PC-3 cells.

These data combined with the shorter-term MTT assays demonstrates asynergistic anti-proliferative effect of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith taxotere pancreatic cancer cells, prostate cancer cells and coloncancer cells.

Example 87 Combination of c-Met Inhibitors and Chemotherapeutic AgentsIn Vitro for the Treatment of Cancer

c-Met is a high affinity receptor for hepatocyte growth factor (HGF)(Weidner K. M. et al. J Cell Biol. 1993 April; 121(1):145-54).Interaction of c-Met with HGF results in autophosphorylation at multipletyrosines, which provide binding sites for and activate severaldownstream signaling components, including Gabl, c-Cbl and PI3 kinase(Bardelli A. et al. Oncogene. 1997 Dec. 18; 15(25):3103-11). Alteredc-Met levels and hyperactivated c-Met have been documented in a varietyof human tumors, including renal, colon and breast cancers and thusc-Met is an attractive cancer therapeutic target (Traxler P. et al. MedRes Rev. 2001 November; 21(6):499-512).(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionehas been shown to inhibit c-Met activity in biochemical assays and in anumber of cell-based assays.(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewas advanced into clinical trials and was well-tolerated, exhibitingsigns of tumor response in late stage cancer patients with metastaticdisease.

To potentially inform phase II clinical trials, an in vitro combinationstudy was initiated using(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith a number of chemotherapeutic agents (gemcitabine, docetaxel andcarboplatin) that are currently in clinical use.

MIA PaCa-2 (also referred to as PACA2), PANC-1, CFPAC-1, Hs766T, DU-145,PC-3 and SK-OV-3 cells were maintained in DMEM supplemented with 10%fetal bovine serum (FBS), penicillin, streptomycin, and fungizone.AsPC-1 and 22Rv-1 cells were maintained in RPMI1640 supplemented with10% fetal bovine serum (FBS), penicillin, streptomycin, and fungizone.HPAF-II cells were maintained in MEM supplemented with 10% fetal bovineserum (FBS), penicillin, streptomycin, and fungizone. For the MTS assay,cells were plated in 96-well plates at 2,000 cells per well andincubated with various doses of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionein combination with gemcitibine, docetaxel or carboplatin for 72 hr. Thelayout of dose range of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione,gemcitibine, docetaxel and carboplatin for drug combination analysis wasdetermined MTS was added to each well and plates were incubated for 4 hrat 37° C. The absorbance of each well was measured at 492 nm using amicroplate reader. Combination indexes of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gemcitibine, docetaxel or carboplatin among the various cell lineswas determined by CalcuSyn™ (Biosoft).

Combination indices of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dionewith gemcitabine, Docetaxel, and carboplatin in various cell lines wereanalyzed.

As described in Example 85, the combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand gemcitabine shows a range of synergistic anti-proliferative effectsin three of five human pancreatic cancer lines.

The combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand Docetaxel demonstrates a range of synergistic anti-proliferativeeffects in two of three human prostate cancer lines, i.e. 22RV1 andDU145 as shown in Table 17.

The combination of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand carboplatin demonstrates a slightly antagonistic effect in theSK-OV-3 ovarian carcinoma cell line.

TABLE 17 Combination Cell Chemotherapeutic Index Values Combination LineTissue Origin Agent at ED₅₀ Conclusion 22RV1 Prostate Docetaxel 0.7Synergism DU-145 Prostate Docetaxel 0.7 Synergism

Example 88 Combination of c-Met Inhibitors and Docetaxel In Vivo for theTreatment of Gastric Cancer

A xenograft model was used to study the effects of a combinatorialtherapy including(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dioneand Docetaxel (DTX) on gastric cancer cells. Specifically, gastriccancer cell lines MKN-45 and Hsc-39 were tested. Evidence of thesynergistic anti-proliferative effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione(Agent A) and Docetaxel (DTX) in a MKN-45 human gastric tumor xenograftmodel are provided in Table 18 and FIG. 9. Similarly, evidence of thesynergistic anti-proliferative effects of(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5,-dione(Agent A) and Docetaxel (DTX) in a Hsc-39 human gastric tumor xenograftmodel are provided in Table 19 and FIG. 10.

TABLE 18 Dosage T/C Min BW loss Mor- (mg/kg) Route Schedule (on day) (g)tality Non- — — qdx5x2 1.00 −1.8 (10) — Treatment Agent A 300 p.o.qdx5x2 0.61 (15) −1.3 (10) 0/5 Docetaxel 15 i.v. single 0.68 (7) −3.8(10) 0/5 Docetaxel 7.5 i.v. single 0.63 (15) −3.0 (4)  0/5 Agent A + 300p.o. qdx5x2 −3.9 (10) 0/5 Docetaxel 15 i.v. single 0.29 (15) Agent A +300 p.o. qdx5x2 −2.7 (4)  0/5 Docetaxel 7.5 i.v. single 0.37 (15)

TABLE 19 Dosage (mg/kg) Route Schedule T/C Min (on day) Non-Treatment —— — 1.00 Agent A 300 p.o. qdx5x2 0.63 (10) Docetaxel 15 i.v. single 0.40(17) Docetaxel 7.5 i.v. single 0.46 (17) Agent A + 300 p.o. qdx5x2 0.19(14) Docetaxel 15 i.v. single Agent A + 300 p.o. qdx5x2 0.40 (17)Docetaxel 7.5 i.v. single

1. A method of treating a cell proliferative disorder, said methodcomprising administering, to a subject in need thereof, atherapeutically effective amount of a composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,or a pharmaceutically acceptable salt thereof, or a prodrug ormetabolite thereof, in combination with a therapeutically effectiveamount of a second anti-proliferative agent, wherein said cellproliferation disorder is treated.
 2. The method of claim 1, wherein thesecond anti-proliferative agent is a kinase inhibitor, an alkylatingagent, an antibiotic, an anti-metabolite, a detoxifying agent, aninterferon, a polyclonal or monoclonal antibody, a HER2 inhibitor, ahistone deacetylase inhibitor, a hormone, a mitotic inhibitor, an MTORinhibitor, a taxane or taxane derivative, an aromatase inhibitor, ananthracycline, a microtubule targeting drug, a topoisomerase poisondrug, or a cytidine analogue drug.
 3. The method of claim 2, wherein thekinase inhibitor is a serine/threonine kinase inhibitor.
 4. The methodof claim 2, wherein the kinase inhibitor is a tyrosine kinase inhibitor.5. The method of claim 2, wherein said kinase inhibitor is sorafenib,sunitinib, erlotinib, imatinib or gefitinib.
 6. The method of claim 2,wherein said alkylating agent is cisplatin or carboplatin.
 7. The methodof claim 2, wherein said anti-metabolite is gemcitabine, fluorouracil,TS-1 or capecitabine.
 8. The method of claim 2, wherein said mitoticinhibitor is camptothecin or irinotecan.
 9. The method of claim 2,wherein said taxane or taxane derivative is paclitaxel or docetaxel. 10.The method of claim 1, wherein said cell proliferative disorder is aprecancerous condition.
 11. The method of claim 1, wherein said cellproliferative disorder is a cancer.
 12. The method of claim 1, whereinsaid cell proliferative disorder is a hematologic tumor or malignancy.13. The method of claim 1, wherein said cell proliferative disorder is asolid tumor (or tumors).
 14. The method of claim 11, wherein said canceris lung cancer, small cell lung cell cancer, non-small cell lung cancer,colon cancer, breast cancer, pancreatic cancer, prostate cancer, renalcancer, cervical cancer, brain cancer, gastric/stomach cancer, uterinecancer, intestinal cancer, hepatic cancer, chronic myelogenous leukemia,melanoma, ovarian cancer, translocation-associated renal cell carcinoma(RCC), alveolar soft part sarcoma (ASPS), clear cell sarcoma (CCS), orhepatocellular carcinoma.
 15. The method of claim 11, wherein saidtreating cancer comprises a reduction in tumor size.
 16. The method ofclaim 11, wherein the cancer is metastatic cancer.
 17. The method ofclaim 11, wherein said treating cancer comprises inhibition ofmetastatic cancer cell invasion.
 18. The method of claim 1, furthercomprising administering radiation therapy.
 19. The method of claim 1,wherein the cells with proliferative disorder contain DNA encodingc-Met.
 20. The method of claim 19, wherein the cells have aconstitutively enhanced c-Met activity.
 21. The method of claim 1,wherein the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered at a dose range between 0.1 mg/day to 10 g/day.
 22. Themethod of claim 21, wherein the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered at a dose range between 0.1 mg/day to 5 g/day.
 23. Themethod of claim 22, wherein the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered at a dose range between 10 mg/day to 1 g/day.
 24. Themethod of claim 23, wherein the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered at a maximal daily dose of 720 mg.
 25. The method ofclaim 24, wherein the(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered at a dose of 360 mg, provided twice a day.
 26. Themethod of claim 1, wherein the composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,and the second anti-proliferative agent are administered intravenously,orally or intraperitoneally.
 27. The method of claim 1, wherein thesecond anti-proliferative agent is administered simultaneously with,preceding administration of, or following administration of thecomposition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione.28. The method of claim 27, wherein the second anti-proliferative agentis administered within 24 hours after the composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dioneis administered.
 29. The method of claim 1, wherein said compositionfurther comprises one or more pharmaceutically acceptable carriers orexcipients.
 30. The method of claim 1, wherein said secondanti-proliferative agent comprises one or more pharmaceuticallyacceptable carriers or excipients.
 31. The method of claim 1, whereinthe subject is a human.
 32. A kit for the treatment of a cellproliferative disorder in a subject comprising separate vials containinga composition comprising(−)-trans-3-(5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl)-4-(1H-indol-3-yl)pyrrolidine-2,5-dione,or a pharmaceutically acceptable salt thereof, or a prodrug ormetabolite thereof, and a second anti-proliferative agent, withinstructions for administering said composition and secondanti-proliferative agent.